Kinetics of reaction of peroxynitrite with selenium- and sulfur-containing compounds: Absolute rate constants and assessment of biological significance

Article abstract of DOI:10.1016/j.freeradbiomed.2015.10.424

Peroxynitrite (the physiological mixture of ONOOH and its anion, ONOO^-) is a powerful biologically-relevant oxidant capable of oxidizing and damaging a range of important targets including sulfides, thiols, lipids, proteins, carbohydrates and nucleic acids. Excessive production of peroxynitrite is associated with several human pathologies including cardiovascular disease, ischemic-reperfusion injury, circulatory shock, inflammation and neurodegeneration. This study demonstrates that low-molecular-mass selenols (RSeH), selenides (RSeR') and to a lesser extent diselenides (RSeSeR') react with peroxynitrite with high rate constants. Low molecular mass selenols react particularly rapidly with peroxynitrite, with second order rate constants k₂ in the range 5.1×10⁵-1.9×10⁶ M^-1 s^-1, and 250-830 fold faster than the corresponding thiols (RSH) and many other endogenous biological targets. Reactions of peroxynitrite with selenides, including selenosugars are approximately 15-fold faster than their sulfur homologs with k₂ approximately 2.5×10³ M^-1 s^-1. The rate constants for diselenides and sulfides were slower with k₂ 0.72-1.3×10³ M^-1 s^-1 and approximately 2.1×10² M^-1 s^-1 respectively. These studies demonstrate that both endogenous and exogenous selenium-containing compounds may modulate peroxynitrite-mediated damage at sites of acute and chronic inflammation, with this being of particular relevance at extracellular sites where the thiol pool is limited.

Full text of DOI:10.1016/j.freeradbiomed.2015.10.424

Free Radical Biology and Medicine 89 (2015) 1049–1056
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Free Radical Biology and Medicine
journal homepage: www.elsevier.com/locate/freeradbiomed
Original Contribution
Kinetics of reaction of peroxynitrite with selenium- and
sulfur-containing compounds: Absolute rate constants and assessment
of biological significance
Corin Storkey a,b, David I. Pattison a,b, Marta T. Ignasiak c, Carl H. Schiesser d,
Michael J. Davies a,b,c,
n
a The Heart Research Institute, 7 Eliza Street, Newtown, NSW 2042, Australia
b Faculty of Medicine, University of Sydney, Sydney, NSW 2006, Australia
c Department of Biomedical Sciences, Panum Institute, University of Copenhagen, Belgdamsvej 3, Copenhagen 2200, Denmark
d School of Chemistry, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010, Australia
a r t i c l e i n f o
a b s t r a c t
Peroxynitrite (the physiological mixture of ONOOH and its anion, ONOOꢀ) is a powerful biologically-
relevant oxidant capable of oxidizing and damaging a range of important targets including sulfides,
thiols, lipids, proteins, carbohydrates and nucleic acids. Excessive production of peroxynitrite is asso-
ciated with several human pathologies including cardiovascular disease, ischemic-reperfusion injury,
circulatory shock, inflammation and neurodegeneration. This study demonstrates that low-molecular-
mass selenols (RSeH), selenides (RSeR') and to a lesser extent diselenides (RSeSeR') react with perox-
ynitrite with high rate constants. Low molecular mass selenols react particularly rapidly with perox-
ynitrite, with second order rate constants k2 in the range 5.1 ꢁ 105–1.9 ꢁ 106 Mꢀ1 sꢀ1, and 250–830 fold
faster than the corresponding thiols (RSH) and many other endogenous biological targets. Reactions of
peroxynitrite with selenides, including selenosugars are approximately 15-fold faster than their sulfur
homologs with k2 approximately 2.5 ꢁ 103 Mꢀ1 sꢀ1. The rate constants for diselenides and sulfides were
slower with k2 0.72–1.3 ꢁ 103 Mꢀ1 sꢀ1 and approximately 2.1 ꢁ 102 Mꢀ1 sꢀ1 respectively. These studies
demonstrate that both endogenous and exogenous selenium-containing compounds may modulate
peroxynitrite-mediated damage at sites of acute and chronic inflammation, with this being of particular
relevance at extracellular sites where the thiol pool is limited.
Article history:
Received 18 June 2015
Received in revised form
26 October 2015
Accepted 28 October 2015
Available online 31 October 2015
Keywords:
Peroxynitrite
Selenium
Selenols
Protein oxidation
Antioxidants
Inflammation
& 2015 Elsevier Inc. All rights reserved.
1. Introduction
two-electron oxidation, or via limited generation of reactive sec-
ꢂ
ꢂ
ondary radicals such as HO and NO2 [16–18]. In the presence of
bicarbonate (HCO3ꢀ) additional reactions involving the peroxyni-
trosocarbonate anion (ONOOCO2ꢀ), and radicals derived from
Peroxynitrous acid (ONOOH) is a potent oxidant and nitrating
species that is formed in vivo and in vitro by the cross-reaction
(termination) of two biologically-important radicals: superoxide
ꢂ
ꢂ
homolysis of this species (HO and CO3 ꢀ) also play a role in in-
ꢂ
(O2 ꢀ; generated by a respiratory burst from activated leukocytes)
ducing oxidative damage [17].
ꢂ
Excessive, mistimed or misplaced production of peroxynitrite
(the physiological mixture of ONOOH and ONOOꢀ) has been as-
sociated with the development of organ damage and dysfunction
in a number of pathologies including cardiovascular disease [19–
21], ischemia-reperfusion injury [22,23], liver damage [24], circu-
latory shock [23], central nervous system damage [25], and neu-
rodegeneration [26,27], amongst others. In such circumstances
endogenous cellular and extracellular defense mechanisms may be
overwhelmed, and synthetic materials that react rapidly with
peroxynitrite may represent novel pharmacological protective
agents for such conditions [28]. A number of low-molecular-mass
compounds have been postulated as biological targets for, or
protective agents against, peroxynitrite-mediated damage
and nitric oxide (NO ; generated by nitric oxide synthase enzymes
present in multiple cells) [1–3]. This species, and to a lesser extent
its anion ONOOꢀ (pKa ꢃ6.8, though this is buffer dependent [4,5]),
oxidizes a wide variety of biomolecules including thioethers (such
as methionine [6]), thiols [7], lipids [8,9], proteins (e.g. [10,11]),
carbohydrates [12] and nucleic acids [13–15] either through direct
Abbreviations: GSH, glutathione; HOX, hypohalous acids; MPO, myeloperoxidase;
Sec, selenocysteine; SeMet, selenomethionine
n
Corresponding author at: Department of Biomedical Sciences, Building 4.5,
Panum Institute, University of Copenhagen, Belgdamsvej 3, Copenhagen 2200,
Denmark.
E-mail address: davies@sund.ku.dk (M.J. Davies).
http://dx.doi.org/10.1016/j.freeradbiomed.2015.10.424
0891-5849/& 2015 Elsevier Inc. All rights reserved.

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C. Storkey et al. / Free Radical Biology and Medicine 89 (2015) 1049–1056
including thiols (e.g. glutathione, GSH), ascorbate, carbon dioxide
(CO2) and synthetic metalloporphyrins [29–32]. Additional studies
have shown that reaction of peroxynitrite with some proteins in-
cluding peroxidases, peroxiredoxins, hemoglobin, albumin and
some selenoproteins is very rapid [29–34]. In the light of the
concentrations of these species present in vivo it has been pos-
tulated that reaction with CO2 (to give the nitrosoperoxycarbonate
anion [35,36]) and GSH is likely to be quantitatively the most
significant [32], though kinetic data for the reactions of perox-
ynitrite particularly with seleno-species are incomplete.
alkaline ONOOꢀ stock solutions.
2.2. Kinetic measurements
Stopped-flow studies were carried out using an Applied Pho-
tophysics SX.18 MV stopped-flow system as described previously
[54,55]. Each stopped-flow measurement was repeated 10 times
with the data averaged to improve the signal to noise ratio.
Rate constants for the reactions of peroxynitrite with the se-
lenol (reduced, RSeH) forms of Sec, selenocystamine, 3-seleno-
propionic acid [IUPAC name: 3-(hydroseleno)propanoic acid], and
selenocysteine methyl ester, were determined by direct kinetics.
The selenols were prepared in situ by reduction of the corre-
sponding diselenide with a 10-fold excess of NaBH4 under an ar-
gon atmosphere, as described previously [55]. The reduced sele-
nols were then diluted with deoxygenated phosphate buffer
(200 mM) to the required concentrations, before reaction with
Selenium, in the form of selenocysteine (Sec), a RNA coded
amino acid, plays a critical role in the activity of a number of
cellular protective enzymes including glutathione peroxidase
(GPx), thioredoxin reductase (TrxR), and some isoforms of me-
thionine sulfoxide reductase [37,38]. GPx in conjunction with its
co-factor, glutathione (GSH), catalyzes the reduction of H2O2 (and
for GPx4, lipid hydroperoxides [39]) and peroxynitrite [40]. The
reactions of GPx and peroxiredoxins (and related species) with
peroxynitrite have been postulated to occur via protein-bound Sec
and Cys residues respectively, with second order rate constants
(k2) in the range 105–107 Mꢀ1 sꢀ1 (e.g. [30,33,34,41–44]). How-
ever it is unclear whether the high reactivity of these residues, and
particularly Sec, is an inherent property of the free amino acid
(and related seleno compounds), or a function of the surrounding
protein. The synthetic selenoamide ebselen, and the natural amino
acid selenomethionine (SeMet) are known to react more rapidly
with peroxynitrite than their sulfur analogs, though k2 for these
low-molecular-mass species are, at least for SeMet, considerably
slower (ꢃ1.5 ꢁ 103 Mꢀ1 sꢀ1 [45]) than for the Sec residue of GPx.
We have recently reported novel selenium-containing carbohy-
drates that react with HOX (X¼Cl, Br, SCN) with high rate con-
stants, and protect both isolated and plasma proteins from hypo-
chlorous acid (HOCl)- and chloramine-mediated damage [46–48]
(reviewed [49]). In the light of the high reactivity of these seleno
compounds with HOCl we hypothesized that low-molecular-mass
selenols, seleno sugars, and diselenides would also react rapidly
with peroxynitrite. The absolute rate constants reported here for
these reactions support this hypothesis. These rate constants and
those for their sulfur analogs obtained for reaction with perox-
ynitrite, are also compared to other oxidants to allow the biolo-
gical significance of these reactions and the role of structure in
determining the reaction kinetics to be assessed.
peroxynitrite (100 M) at 22 °C under a N2 atmosphere, with ab-
μ
sorption changes determined at 245 nm (Sec, selenocystamine,
and selenocysteine methyl ester) or 248 nm (3-selenopropionic
acid). At these wavelengths peroxynitrite does not absorb to a
significant extent, thus the selenol concentration for each kinetic
run (50–385 M) was determined by extrapolating the data to
μ
obtain the initial absorbance value (t¼0 s) and converting to
concentration using the corresponding molar absorption coeffi-
cients [55]). In the absence of peroxynitrite the selenols undergo
slow autoxidation under the conditions employed [55], thus in
alternative determinations of these rate constants, the absorption
spectrum of the stock solutions of the selenol was measured be-
fore and after use, with the mean value of the two measurements
used to estimate the selenol concentration.
As the kinetic traces obtained for the reaction of selenols with
peroxynitrite were not always obtained under pseudo first-order
conditions (i.e. with substrate excesses greater than 5-fold over
oxidant), the decay profiles were analyzed using second-order
decay profiles using Pro-KIV (Version 1.0.4.0; Applied Photo-
physics). In order to fit these traces a simple model was used
comprising of two reactions: (i) RSeHþperoxynitrite - product,
kRSeH and (ii) peroxynitrite - decay products, kdecomp. The initial
reactant concentrations were defined (following determination as
described above) and the value of kdecomp was fixed as 0.27 sꢀ1, as
determined from experiments with peroxynitrite in the absence of
selenols (see Supplementary Fig. 1a). This value of kdecomp gives a
rate constant for isomerization of peroxynitrite (kisomerization) of
ꢃ1.3 s ꢀ1 at pH 7.4 and 22 °C in 100 mM phosphate buffer (where
the pKa of peroxynitrite is ꢃ6.8 [4,56]) using the equation re-
ported by Molina et al. [57]. This value is slightly higher than that
reported previously (1.1 sꢀ1) for highly purified peroxynitrite so-
lutions [57]. In order to further reduce the number of fitting
parameters, the peroxynitrite and peroxynitrite decay products
were defined as non-absorbing species at the wavelengths em-
ployed. The model was then allowed to vary the value of kRSeH to
obtain the best fit to the data (Supplementary Fig. 1b and c shows
example data and fits), which was typically achieved within 10–15
iterations. The kRSeH values obtained for each kinetic trace (n46)
were then averaged to obtain the k2 values for each selenol, with
errors reported as 95% confidence limits.
2. Materials and methods
2.1. Materials
3,3′-Diselenodipropionic acid was synthesized and purified as
described previously [50]. The selenosugars were synthesized as
described previously [46,47]. The diselenide of selenocysteine
methyl ester [-SeCH2CH(NH2)C(¼O)OCH3]2 was synthesized as
reported previously [51]. All other chemicals were from Sigma-
Aldrich/Fluka and used without further purification. All selenium-
containing compounds were dissolved in Chelex-treated sodium
phosphate buffer (pH 7.4, 200 mM) prepared using Milli Q water.
Peroxynitrite was prepared as described previously and stored in
0.1 M NaOH (pH ca. 12) [52]. Concentrations of the stock perox-
ynitrite solutions were determined prior to use by spectro-
photometric quantification of its anion form (ONOOꢀ) at high pH
The reactions of peroxynitrite (100 M) with selenides (RSeR',
μ
0.5–1.25 mM), diselenides (RSeSeR', 0.5–1.25 mM), sulfides (RSR',
2.5–12.5 mM) and disulfides (RSSR', 2.5–12.5 mM) were de-
termined by direct kinetic measurements of the loss of peroxyni-
trite measured at 302 nm. As higher concentrations were used for
these substrates, the kinetic data could all be fitted to single ex-
ponential decays using Pro-Data viewer 4.0 (Applied Photophysics)
and OriginPro 7.0 (OriginLab) software. For each substrate the re-
sulting pseudo first order rate constant (kobs) was plotted against
using
mediately before use, peroxynitrite was diluted with MilliQ water
to give 200
M ONOOꢀ in 5 mM NaOH. The strong buffering ca-
a previously reported extinction coefficient [53]. Im-
μ
pacity (200 mM phosphate buffer) of the solutions containing the
selenium compounds prevented changes in pH on mixing (1:1,
resulting in a final buffer concentration of 100 mM) with the

C. Storkey et al. / Free Radical Biology and Medicine 89 (2015) 1049–1056
1051
substrate concentration and the second order (bimolecular) rate
3. Results
constant k2 was obtained from a linear fit of the data (with the
intercept fixed as 0.27 sꢀ1, consistent with the natural decay of
peroxynitrite measured in these studies). All rate constants re-
ported for selenides, diselenides, sulfides and disulfides were de-
rived from 43 independent experiments employing at least four
different substrate concentrations and errors are specified as 95%
confidence limits.
3.1. Determination of rate constants for reaction of peroxynitrite
with low-molecular-mass selenols
Selenols (RSeH) are structurally similar to thiols (RSH), but have
lower pKa values (i.e. are stronger acids) and exist primarily in their
anion form (RSeꢀ, pKa 5.2 for Sec) at neutral pH [58]. Selenols are
Table 1
Summary of rate constants, k2, determined for reaction of peroxynitrite with seleno- and compounds using stopped-flow methods with absorbance detection. Reactions
were carried out in 0.1 M phosphate buffer, pH 7.4 at 22 °C. Second-order rate constants are given with 95% confidence limits. Literature data for the corresponding sulfur-
analogues are provided for comparison.
Substrate
Structure
k2 / Mꢀ1sꢀ1
k2 / Mꢀ1sꢀ1 (for sulfur analogue)
a
b
Selenocysteine (Sec)
(6.670.3) ꢁ 105
(2.670.1) ꢁ 103
a
b
Sec methyl ester
(1.270.7) ꢁ 106
(3.970.1) ꢁ 103
a
c
b
Selenocysteamine
(1.971.0) ꢁ 106
(7.170.4) ꢁ 105
(2.370.1) ꢁ 103
d
e
3-Selenopropionic acid
Selenomethionine (SeMet)
(5.170.7) ꢁ 105
ND
b
b
(2.570.1) ꢁ 103
(1.670.1) ꢁ 102
1.48 ꢁ 103 (at 25 °C [45])
[(1.770.1) ꢁ 103 for acid, and (8.670.2) for anion [6]
b
e
(Se-methyl)-selenocysteine
Selenotalose (SeTal)
(1.970.1) ꢁ 103
ND
b
e
(2.570.1) ꢁ 103
ND
b
b
Selenogulose (SeGul)
(2.570.1) ꢁ 103
(2.170.1) ꢁ 102
b
e
Sec methyl ester diselenide
(7.370.3) ꢁ 102
ND

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C. Storkey et al. / Free Radical Biology and Medicine 89 (2015) 1049–1056
Table 1 (continued )
Substrate
Structure
k2 / Mꢀ1sꢀ1
k2 / Mꢀ1sꢀ1 (for sulfur analogue)
b
b
e
3,3′-Diseleno-dipropionic acid
(1.370.1) ꢁ 103
ND
e
2,2′-diseleno-cystamine
(7.270.4) ꢁ 102
ND
a
Absorbance changes measured at 243 nm to monitor loss of selenol, with extinction coefficient used to calculate selenol concentration.
Absorbance changes measured at 302 nm to monitor loss of ONOOꢀ
Value determined using absorption spectra of selenol before and after kinetic runs.
Absorbance changes measured at 248 nm to monitor loss of selenol, with extinction coefficient used to calculate selenol concentration.
ND, not determined.
b
c
.
d
e
powerful nucleophiles and undergo ready oxidation in air [58], so
these materials were synthesized in their diselenide (RSeSeR') forms.
The selenols were obtained from the diselenides for kinetic studies
by in situ reduction with sodium borohydride [55]. The slow decay of
the stock selenol solutions was accounted for using two different
approaches (see Section 2), which gave k2 values of similar magni-
tude. The rate constants for reaction with peroxynitrite were de-
termined under an atmosphere of N2 by direct stopped-flow meth-
3.2. Determination of rate constants for reaction of peroxynitrite
with low-molecular-mass selenides, diselenides and sulfur analogs
Unlike selenols that have a strong absorbance band in the UV
region (o320 nm), selenides (selenoethers, RSeR'), diselenides
(RSeSeR'), thiols (RSH), thioethers (RSR') and disulfides (RSSR') do
not have significant confounding UV–visible absorbance bands at
the wavelengths examined. As a consequence the kinetics of these
reactions were examined by quantifying the enhanced rate of
decay of peroxynitrite at 302 nm in the presence of these chal-
cogen compounds. Selenoethers, thiols or diselenides (0.5–
ods using final concentrations of 50–385
μ
M selenol and 100 M
μ
peroxynitrite at 22 °C over a period of 0–0.2 s, with changes in ab-
sorbance from the selenol monitored at its λmax [55]. Control ex-
periments with decomposed peroxynitrite in buffer confirmed that
decay products from the oxidant had minimal impact on the selenol
reaction data. The observed absorbance changes on mixing perox-
ynitrite and selenols were fitted directly to second-order kinetics (for
example data and fits see Supplementary Fig. 1b and c) to obtain an
estimate of the second-order rate constants (k2) at each selenol
concentration; these values were then averaged and the 95% con-
fidence intervals were calculated (Table 1).
1.25 mM) were reacted with 100
period of 0–5 s, with changes in absorbance measured at 302 nm.
Thioethers or disulfides (2.5–12.5 mM) were reacted with 100
μM peroxynitrite at 22 °C over a
μ
M
peroxynitrite in a similar manner but over a period of 0–8 s.
Control experiments in the absence of substrate showed that the
spontaneous decay of peroxynitrite at pH 7.4 was less rapid
(Supplementary Fig. 1a; k 0.27 sꢀ1), and not a confounding reac-
tion over these timescales. Absorbance changes were fitted to a
single-exponential function to give kobs (Fig. 1) with subsequent
Fig. 2. Plots of observed rate constants (kobs) against scavenger concentrations for a
range of selenides, diselenides and glutathione (GSH). The second order rate con-
stants, k2, for reaction with peroxynitrite were established from the gradients of
these linear plots. Error bars represent7SEM (n¼3). The y-intercept was fixed as
0.27 sꢀ1, the experimentally-determined rate constant for decay of peroxynitrite in
the absence of added compounds (see text and Supplementary Fig. 1).
Fig. 1. Representative stopped-flow kinetic traces for the reaction of SeTal (0.5–
1.25 mM) with peroxynitrite (0.1 mM) at 22 °C in 0.1 M phosphate buffer (pH 7.4).
The kinetic data obtained at 302 nm are represented by averaging 10 experiments
with the observed rate constant (kobs) generated from the exponential fitted to a
single continuous decay curve.

C. Storkey et al. / Free Radical Biology and Medicine 89 (2015) 1049–1056
1053
plots of kobs against substrate concentration, giving a straight line
(Fig. 2) from which k2 was obtained from the gradient (Table 1).
Kinetic data for 3,3′-diselenocystine could not be acquired due to
its low solubility under the conditions employed and the re-
quirement for high substrate excesses.
molecular-mass thiols (Cys, GSH) or SeMet have been reported to
be in the range 1.4–2.0 ꢁ 104 Mꢀ1 sꢀ1 [7,63,64]. Reaction with
thioethers such as Met has k2 ꢃ1.7 ꢁ 103 Mꢀ1 sꢀ1 [6]). In the light
of these, and previous data [55], it was hypothesized that reaction
of peroxynitrite with selenols should be significantly more rapid
than with analogous thiols, selenides and sulfides, as selenols are
predominantly ionized at neutral pH values (cf. pKa's of 5.2 and
8.4 for Sec and Cys respectively [58,65] and are more nucleophilic
than sulfur centers. However it should be noted that the reactivity
of these compounds is unlikely to be determined solely by pKa
values. Thus it has been reported that changing the pKa value of a
Cys residue from 8.4 to 5 only changes the reactivity by a max-
imum of 10-fold at pH 7.4 as the thiolate availability increases
from ꢃ10% to 100% [66], and many proteins with low pKa Cys
residues react slowly with peroxides or peroxynitrite. The situa-
tion with glutathione peroxidases (GPxs) and peroxiredoxins
(Prxs) is however different, as these are specialized peroxidases
that have evolved to maximize peroxide and peroxynitrite re-
duction [33].
4. Discussion
Peroxynitrite is a major oxidant generated at sites of in-
flammation that is both bactericidal and potentially damaging to
host tissues (reviewed: [16,17,59]). Peroxynitrite can react directly
and rapidly, with proteins, metal ions, selenium-containing pro-
teins and pharmaceuticals, and (via its anion form ONOOꢀ) with
ꢀ
CO2 to give the peroxynitrosocarbonate anion (ONOOCO2
[11,17,59,60]. Homolytic cleavage of both ONOOH and ONOOCO2
)
ꢀ
can result in radical formation and additional damage [17,59,61],
though this homolysis reaction occurs to only a very limited ex-
tent, and its quantitative significance is unclear [18].
The data presented here are consistent with much greater re-
activity of the selenium-containing species compared to their
sulfur analogs. The values of k2 for reaction of peroxynitrite with
selenols are between 250 and 830-fold higher than for the cor-
responding thiols, and up to 2600-fold higher than for the corre-
sponding (oxidized) diselenides under identical conditions (Ta-
ble 1). Selenols also react approximately 500-fold faster than se-
lenides (selenoethers, e.g. SeMet), and up to 5500-fold faster than
sulfides (thioethers, e.g. Met). The value for k2 determined here for
free Sec is lower than that for the active site Sec of GPx (Table 2),
consistent with the hypothesis that the protein structure
The reactivity of peroxynitrite with common amino acids and
the Sec residue of GPx has been examined previously [11,30]. Cys,
Met and Trp are the only free amino acids that undergo appreci-
able direct reaction with peroxynitrite, but other amino acids (e.g.
Tyr, His and Lys) can be modified via secondary radicals, or species
generated in the presence of transition metal ions [10,59]. The rate
constants for reaction of peroxynitrite with the Sec residue of GPx,
and Cys residues of peroxiredoxins, are some of the highest known
for peroxynitrite with k2 in the range ꢃ105– 7 ꢁ 107 Mꢀ1 sꢀ1, with
peroxiredoxins reported to be major targets for this oxidant
[30,41,43,44,62]. In contrast, k2 values for reaction with low-
Table 2
Comparison of second order rate constants, k2, for selected reactions of biologically-relevant oxidants with seleno- and sulfur- compounds at pH 7.4 and ꢃ22 °C unless
indicated otherwise. Data are from the current study unless otherwise noted. For information on the associated errors see Table 1 and cited literature.
Substrate
Cysteine
k (HOCl) (Mꢀ1 sꢀ1
)
k (HOSCN) (Mꢀ1 sꢀ1
)
k (ONOOH) (Mꢀ1 sꢀ1
)
3.1 ꢁ 108 [84]
7.8 ꢁ 104 [54]
2.6 ꢁ 103
4.5 ꢁ 103 (at 37 °C [7])
For Arrhenius plot and further data [5,85]
a
Selenocysteine
N-Ac-cysteine
Cystamine
ND
1.2 ꢁ 106 [55]
7.3 ꢁ 103 [54]
7.8 ꢁ 104 [54]
5.8 ꢁ 106 [55]
6.6 ꢁ 105
2.9 ꢁ 107 [84]
4.15 ꢁ 102 (at 37 °C [63])
2.3 ꢁ 103
ND
ND
a
Selenocystamine
1.9 ꢁ 106
b
7.1 ꢁ 105
GSH
1.1 ꢁ 108 [84]
3.4 ꢁ 107 [84]
2.5 ꢁ 104 [54]
7.3 ꢁ 102
8.0 ꢁ 104 (at 25 °C [86])
o103 [54]
1.36 ꢁ 103 (at 37 °C [5,63])
Methionine
1.6 ꢁ 102
1.7 ꢁ 103 for ONOOH, and 8.6 for ONOOꢀ [6]
3.64 ꢁ 102 (at 37 °C [87])
Selenomethionine
3.2 ꢁ 108 [47]
2.8 ꢁ 103 [55]
2.5 ꢁ 103
1.48 ꢁ 103 (at 25 °C [45])
Selenotalose
Selenogulose
Ebselen
1.0 ꢁ 108 [47]
9.4 ꢁ 107 [47]
ND
ND
ND
2.5 ꢁ 103
2.5 ꢁ 103
3 ꢁ 101 [55]
2 ꢁ 106 (at 25 °C, pH48) [88]
9.7 ꢁ 103 (for human serum albumin at 37 °C [87])
3.8 ꢁ 103 (for Cys residue of human serum albumin at 37 °C [87])
8 ꢁ 106 [30]
Serum albumin
ND
7.6 ꢁ 104 (for bovine serum albumin [54])
Glutathione peroxidase ND
5 ꢁ 105 [55]
1.8 ꢁ 105 (at 37 °C, pH 7.1 [89])
7.4 ꢁ 105 (for oxidized tetramer; [30])
ꢃ106 (for poplar thioredoxin-dependent glutathione peroxidase 5
[90])
c
Peroxiredoxin family
ND
7 ꢁ 107 for human peroxiredoxin 5 [41,62]
1.5 ꢁ 106 for AhpC (from Salmonella typhimurium [43])
3.0 ꢁ 103 (for oxidized AhpC [43])
ꢃ105 for thioredoxin peroxidases I/II (from Saccharomyces cerevisiae
[44])
ND: not determined.
a
Value determined using extinction coefficient to calculate selenol concentration.
b
c
Value determined using absorption spectra of selenol before and after kinetic runs.
Data for peroxiredoxins not determined; for other Cys-containing proteins k2 is 1.0–7.6 ꢁ 104 Mꢀ1 sꢀ1 [54].

1054
C. Storkey et al. / Free Radical Biology and Medicine 89 (2015) 1049–1056
modulates the environment and reactivity of the selenol. This
conclusion is supported by the variation in k2 between the various
selenols (Table 1), with the data being consistent with the
neighboring groups modulating selenol reactivity. Of the selenols
examined, the lowest k2 values were for 3-selenopropionic acid
and Sec (both approximately 5 ꢁ 105 Mꢀ1 sꢀ1) with higher values
determined for Sec methyl ester (k2 1.2 ꢁ 106 Mꢀ1 sꢀ1) and re-
reactions. These reactions also generate nitroso, nitro, nitroso-thiol
and nitro-thiol products, in addition to the disulfides [68–72]. The
identities of the materials formed from the seleno species remain
to be determined, though it is probable that the major route is a
2-electron (molecular) reaction to give a selenenic (RSeOH) spe-
cies before reaction with another selenol, to give the diselenide.
The data reported in Table 1 allow conclusions to be drawn as
to the potential importance of peroxynitrite reactions with sulfur-
versus seleno-species within cells and extracellular fluids. Within
cells, the high concentration of low-molecular-mass thiols (e.g.
GSH; 2–5 mM [73]) compared to sub micromolar levels of en-
dogenous low-molecular-mass selenols/selenides/diselenides,
suggests that there will be a marked selectivity for thiol oxidation,
despite the higher reactivity of selenols/selenides/diselenides. In
duced selenocystamine (k2 7.1–19.0 ꢁ 105 Mꢀ1 sꢀ1).
A similar
trend is apparent for the corresponding thiols, though these values
were much lower than for the selenium analogs, with the highest
values determined for the methyl ester of cysteine
(3.9 ꢁ 103 Mꢀ1 sꢀ1). These data suggest that the presence of a
suitably positioned ionized carboxyl group decreases the rate of
reaction, and a protonated amine group enhances the rate of re-
action, though the exact reason for this observation remains to be
established.
Previous studies have reported k2 for reaction of peroxynitrite
with Cys to be 4.5 ꢁ 103 Mꢀ1 sꢀ1 [7] and 1.36 ꢁ 103 Mꢀ1 sꢀ1 at
37 °C for GSH [63]. These values are between 1.7 and 1.9-fold faster
than determined here at 22 °C. The similarity of the ratio of the k2
values at these two different temperatures for Cys and GSH, sug-
gests that realistic estimates can be made for other species at
37 °C, if required.
All the selenides examined had similar k2 values, with both the
novel 6-membered ring seleno-sugar SeGul, and the 5-membered
ring species SeTal, being 2.5 ꢁ 103 Mꢀ1 sꢀ1. Interestingly 3-(sele-
nomethyl)cysteine (1.9 ꢁ 103 Mꢀ1 sꢀ1) displayed a slightly lower
rate constant than SeMet (2.4 ꢁ 103 Mꢀ1 sꢀ1), suggesting that an
increased distance between the seleno center and the other
functional groups enhances reactivity; this may reflect a diminu-
tion of through bond electron withdrawing effects. Comparison of
these data with those of the corresponding sulfur-analogs (e.g.
thiogulose, SGul, k2 2.1 ꢁ 102 Mꢀ1 sꢀ1) indicates an approximate
12-fold decrease in reactivity, consistent with the difference be-
tween SeMet and Met (Table 1).
the case of exogenously added species (supplementation), high M
μ
levels of added seleno compounds would be needed before direct
seleno oxidation becomes significant.
With Sec residues on proteins, the situation is more favorable.
As thioredoxin reductase is only present at nM levels in cells [74]
this is unlikely to be a major target when compared to GSH or
protein thiols. In contrast, cellular concentrations of GPx are high
(ꢃ2
μM [74,75]) and may compete with GSH for peroxynitrite in
the light of the values presented in Table 2. These data are con-
sistent with a report that GPx can act as a peroxynitrite reductase,
thereby preventing peroxynitrite-induced damage [76]. A similar
argument applies to intracellular protein-bound Cys residues,
which are present at millimolar concentrations (cf. protein thiol
concentrations in mitochondria of 60–90 mM [77,78]), though the
higher k2 values for some protein-Cys residues compared to GSH
(cf. Table 2), may mitigate against this. In particular, proteins with
low pKa Cys residues (e.g. cytosolic peroxiredoxins, Prx1 and Prx2,
which are present in Jurkat cells at ꢃ65
μ
M, and mitochondrial
M [79]) would be expected to out-
Prx3 which is present at ꢃ125
μ
compete reaction with GPx.
In extracellular fluids, the situation is very different, as the
For the diselenides the highest values of k2 were determined for
3,3′-diselenodipropionoic acid (1.3 ꢁ 103 Mꢀ1 sꢀ1) with lower values
determined for the 3,3′-diselenide form of the Sec methyl ester
(7.3 ꢁ 102 Mꢀ1 sꢀ1) and 3,3′-diselenocystamine (7.2 ꢁ 102 Mꢀ1 sꢀ1).
These values indicate that diselenides per se offer limited scavenging
activity against peroxynitrite in biological systems when compared to
their reduced (selenol) forms. However as diselenides are likely to be
reduced in vivo (cf. the ready reduction of disulfides to thiols), these
species may act as potential pro-forms of the more reactive selenols.
Data for the analogous disulfide reaction could not be obtained due to
the (relatively) rapid intrinsic decay of peroxynitrite under these
conditions, consistent with a previous report for glutathione disulfide
where no increased rate of peroxynitrite decay was observed [63]. The
cyclic disulfide lipoic acid does however enhance the rate of perox-
ynitrite decay (k2 1.4 ꢁ 103 Mꢀ1 sꢀ1 at 37 °C), with this resulting in
disulfide S-oxide (thiosulfinate) formation [63].
concentration of the low-molecular-mass thiol pool in plasma is
low (cf. 12–19
incorporated Cys (primarily Cys-34 of human serum albumin;
nominally 400–600 M, although a significant fraction of this is
μ
M) [80], compared to the concentration of protein-
μ
present in modified forms [80]). At these thiol concentrations, and
with a 250–830 greater reactivity of selenols over thiols with
peroxynitrite (Table 1), reaction of peroxynitrite with low-mole-
cular-mass plasma selenols, selenides and diselenides would be
competitive if these were present at low
μM levels. For extra-
cellular selenoproteins, the (up to) 10 Sec residues of selenoprotein
P (the most abundant seleno species in plasma [81]) would be
expected to be competitive for peroxynitrite (relative to both free-
and protein Cys residues) on the basis of the determined k2 value
for free Sec (Tables 1 and 2), and the reported plasma concentra-
tions of this seleno protein, which range from 0.8 to 2
μ
M, and
hence a total Sec concentration of up to 20 M [81]. This conclu-
μ
Although the present study has provided absolute kinetic data
for the reactivity of peroxynitrite with selenols and other seleno-
species, the mechanisms and products of these reactions remain to
be fully defined. The major pathway for the analogous thiol reac-
tions has been reported to be a (2-electron) molecular process
involving nucleophilic attack of the thiolate anion on one of the
peroxidic oxygens of peroxynitrite resulting in the formation of an
intermediate sulfenic acid (RSOH), which then reacts rapidly with
another thiol to give the disulfide [7]. The RSOH intermediate has
been detected in some cases (e.g. on peroxiredoxins [43] and al-
bumin [67]). However radicals that may be generated as a result of
peroxynitrite homolysis (either in the absence or presence of CO2)
sion is consistent with experimental data [29]. Plasma con-
centrations of extracellular GPx (GPx3) and thioredoxin reductase
are lower: 0.24–0.6
μ
M (and hence 0.96–2.4 M Sec given its
μ
tetrameric structure) and 0.3 nM respectively [74, 82]. On the basis
of these data, reaction of peroxynitrite with the Sec residues of
GPx3 may be significant (given a concentration ratio of protein Sec
to Cys of 0.96–2.4
Table 2), but scavenging of peroxynitrite by extracellular thior-
edoxin reductase would not.
μ
M : 400–600
μM, and a k2 ratio of ꢃ800; cf.
Overall, these data indicate that selenols and other seleno-
species are targets for peroxynitrite, with rate constants for sele-
nols being significantly faster than for thiols, and selenides being
faster than sulfides/thioethers. Diselenides also exhibit consider-
able reactivity towards peroxynitrite, though their k2 values are
lower than for both selenols and selenides. The diselenides are
ꢂ
may also mediate thiol oxidation via formation of thiyl (RS ),
disulfide radical-anion (RSSR ꢀ) and sulfinyl (RSO ) radicals, with
ꢂ
ꢂ
these and other species participating in dioxygen-dependent chain

C. Storkey et al. / Free Radical Biology and Medicine 89 (2015) 1049–1056
1055
however more reactive than most disulfides, which react very
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HOCl and hypothiocyanous acid (HOSCN), with these targets. Thus
HOCl reacts with thiols approximately five orders of magnitude faster
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generated by both neutrophil- and macrophage-derived processes
being important in both pathogen removal, and host tissue damage,
with these mechanisms being both complementary and redundant
(i.e. capable of replacing each other, at least to a major extent).
Acknowledgments
We are grateful to the Australian Research Council (through the
Centres of Excellence Scheme, CE0561607, and Discovery Pro-
grams DP0988311), the National Heart Foundation of Australia
(Grants-in-aids G09S4313 and G11S 5811) and the Novo Nordisk
Foundation (Laureate Research Grant NNF13OC0004294 to MJD)
for financial support. We thank Professors Vimal K. Jain and K.
Indira Priyadarsini of Bhabha Atomic Research Centre, Mumbai
(India), for providing 3,3′-diselenodipropionic acid.
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