The use of cyclic nitroxide radicals as HNO scavengers

Article abstract of DOI:10.1016/j.jinorgbio.2012.10.002

Reduction of cyclic stable nitroxides (RNO) by HNO to the respective hydroxylamines (RNO-H) has been demonstrated using EPR spectrometry. HNO shows low reactivity toward piperidine, pyrrolidine and nitronyl nitroxides with rate constants below 1.4 × 10⁵ M^{- 1} s^{- 1} at pH 7.0, despite the high driving force for these reactions. The rate constants can be predicted assuming that the reactions take place via a concerted proton-electron transfer pathway and significantly low self-exchange rate constants for HNO/NO and RNO-H/RNO. NO does not react with piperidine and pyrrolidine nitroxides, but does add to HNO forming the highly oxidizing and moderately reducing hyponitrite radicals. In this work, the radicals are produced by pulse radiolysis and the rate constants of their reactions with 2,2,6,6,-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl (TEMPOL) and 3-carbamoyl-PROXYL have been determined at pH 6.8 to be (2.4 ± 0.2) × 10⁶, (9.8 ± 0.2) × 10⁵, (5.9 ± 0.5) × 10⁵ M^{- 1} s ^{- 1}, respectively. This low reactivity implies that NO competes efficiently with these nitroxides for the hyponitrite radical. The ability of TEMPOL and 2-(4-carboxyphenyl)-4,4,5,5,-tetramethyl-imidazoline-1-oxyl-3-oxide (C-PTIO) to oxidize HNO and their different reactivity toward NO are used to quantify HNO formed via acetohydroxamic acid oxidation. The extent of TEMPOL or C-PTIO reduction was similar to the yield of HNO formed upon oxidation by OH under anoxia, but not by the metmyoglobin and H₂O₂ reaction system where both nitroxides catalytically facilitate H ₂O₂ depletion and nitrite accumulation. In this system the conversion of C-PTIO into 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1- oxyl (C-PTI) is a minor reaction, which does not provide any mechanistic insight.

Full text of DOI:10.1016/j.jinorgbio.2012.10.002

Journal of Inorganic Biochemistry 118 (2013) 155–161
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
The use of cyclic nitroxide radicals as HNO scavengers
a
b
c,
Yuval Samuni , Uri Samuni , Sara Goldstein ⁎
a
Oral and Maxillofacial Surgery, The Barzilai Medical Center, Ashkelon, Israel
Chemistry and Biochemistry Department, Queens College, City University of New York, Flushing, NY 11367, USA
Institute of Chemistry, The Accelerator Laboratory, the Hebrew University of Jerusalem, Jerusalem 91904, Israel
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 May 2012
Received in revised form 11 September 2012
Accepted 4 October 2012
Available online 12 October 2012
Reduction of cyclic stable nitroxides (RNO) by HNO to the respective hydroxylamines (RNO-H) has been demon-
strated using EPR spectrometry. HNO shows low reactivity toward piperidine, pyrrolidine and nitronyl nitroxides
with rate constants below 1.4×105
M
−1 −1
s
at pH 7.0, despite the high driving force for these reactions. The rate
constants can be predicted assuming that the reactions take place via a concerted proton–electron transfer path-
way and significantly low self-exchange rate constants for HNO/NO and RNO-H/RNO. NO does not react with
piperidine and pyrrolidine nitroxides, but does add to HNO forming the highly oxidizing and moderately reducing
hyponitrite radicals. In this work, the radicals are produced by pulse radiolysis and the rate constants of their
reactions with 2,2,6,6,-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl
Keywords:
Hydrogen atom transfer
Hydroxylamine
Hyponitrite radical
EPR
Acetohydroxamic acid
Radiolysis
6
5
(TEMPOL) and 3-carbamoyl-PROXYL have been determined at pH 6.8 to be (2.4± 0.2)×10 , (9.8± 0.2)×10 ,
(5.9± 0.5)×105
M
−1
s
−1
, respectively. This low reactivity implies that NO competes efficiently with these
nitroxides for the hyponitrite radical. The ability of TEMPOL and 2-(4-carboxyphenyl)-4,4,5,5,-tetramethyl-
imidazoline-1-oxyl-3-oxide (C-PTIO) to oxidize HNO and their different reactivity toward NO are used to quantify
HNO formed via acetohydroxamic acid oxidation. The extent of TEMPOL or C-PTIO reduction was similar to the
•
yield of HNO formed upon oxidation by OH under anoxia, but not by the metmyoglobin and H
O
2 2
reaction system
where both nitroxides catalytically facilitate H
O
2 2
depletion and nitrite accumulation. In this system the conver-
sion of C-PTIO into 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl (C-PTI) is a minor reaction,
which does not provide any mechanistic insight.
©
2012 Elsevier Inc. All rights reserved.
1
. Introduction
oxyl 3-oxide (PTIO) and 2-(4-carboxyphenyl)-4,4,5,5,-tetramethyl-
imidazoline-1-oxyl-3-oxide (C-PTIO), and have shown unequivocally
that HNO reduces these nitroxides to their respective hydroxylamines
(reaction (1)) [6,7].
Cyclic stable nitroxide radicals (RNO), also known as aminoxyl or
nitroxyl radicals, have been used for many years as biophysical probes
to monitor membrane dynamics, cellular pH and O level [1–3]. RNO
2
can shuttle through one-electron redox reactions among three oxidation
states as shown in Scheme 1 for piperidine nitroxides. The rate constant
of HNO reaction with the stable cyclic nitroxide 4-hydroxy-2,2,6,6-
tetramethyl piperidine-1-oxyl (TEMPOL) has been determined to be
RNO þ HNO → RNOꢀH þ NO
ð1Þ
Most RNO do not react with NO, which readily adds to HNO
forming the hyponitrite radical HN O (reaction (2)) [8–10].
2 2
×104
M
−1
s
−1
using the HNO donor Angeli's salt (AS) and competi-
•
8
III
tion kinetics against metmyoglobin (MbFe ) assuming that TEMPOL
oxidizes HNO [4]. Surprisingly, its structural analog 2,2,6,6-tetramethyl
piperidine-1-oxyl (TEMPO) has been reported to reduce HNO, and
there is neither experimental evidence nor any explanation on how
the authors arrived at this conclusion [5]. We have previously studied
using AS and EPR spectrometry the reactions of HNO with TEMPOL,
the nitronyl nitroxides 2-phenyl-4,4,5,5-tetramethylimidazoline-1-
ꢁ
6
−1 −1
HNO þ NO → HN O
k ¼ 5:8 ꢂ 10 M
s
ð2Þ
2
2
2
•
•−
2
HN O
2
/N
2
O
2
a
(pK =5.5) are strongly oxidizing (1.75 V/0.96 V)
and moderately reducing (−0.06 V/−0.38 V) radicals [11], which at
neutral solutions have sufficient driving force to oxidize as well as
reduce RNO [12–17].
In contrast to piperidine and pyrrolidine nitroxides, which do not
react with NO, nitronyl nitroxides react uniquely with NO forming
the respective imino nitroxides as demonstrated below for C-PTIO
⁎
Corresponding author. Tel.: +972 2 6586478; fax: +972 2 6586925.
E-mail address: sarag@vms.huji.ac.il (S. Goldstein).
0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2012.10.002

156
Y. Samuni et al. / Journal of Inorganic Biochemistry 118 (2013) 155–161
Scheme 1. Oxidation and reduction of piperidine nitroxides (R _ H, TEMPO; R _ OH, TEMPOL).
(
reaction (3), k
3
=1×104 M−1 s−1 [18,19]). Both C-PTIO and C-PTI
the commercial salt. For pulse radiolysis experiments, solutions of sodi-
being nitroxides are reducible by HNO [6].
um trans-hyponitrite (4 mM) were prepared by adding the salt to
2
N O-saturated solutions containing 40 mM phosphate buffer (pH 6.8),
and were used within 1 h.
III
Metmyoglobin (MbFe ) was prepared by adding excess of ferricya-
nide to myoglobin in 10 mM phosphate buffer (PB) at pH 7.0 followed
by chromatographic separation through a Sephadex G-25 column. The
III
concentration of MbFe was determined spectrophotometrically using
−
1
cm−1 [32]. N
ε
408 =188,000 M
from traces of O
Alltech Associate Inc.).
2
O gas (Maxima, Israel) was purified
2
by passing it through an oxygen trap (OXY-TRAP,
ð3Þ
Since RNO has a characteristic EPR spectrum whereas RNO-H is
diamagnetic and EPR-silent, the reduction of RNO by HNO can be mon-
itored using EPR spectroscopy. The characteristic EPR spectra of nitronyl
nitroxides and imino nitroxides are distinctly different, thus enabling
the detection and in some cases quantification of NO by monitoring
the conversion of nitronyl nitroxide to imino nitroxide [18–21].
2
.2. Analysis
Nitrite formation was assayed by mixing equal volumes of the sam-
ple and the Griess reagent. Analysis of nitrite produced under anoxia
was done as follows. 1.5 mL of the reagent was placed in a 4 mL optical
cell sealed with a rubber septum. The reagent was deoxygenated by
a
HNO (pK =11.4 [22,23]) has been demonstrated as a unique spe-
passing N
.5 mL of the anoxic sample through the rubber septum. The absorption
at 540 nm was read 15 min after the addition of the sample. Calibration
curves were prepared using known concentrations of nitrite. H con-
centration was determined by the molybdate-activated iodide assay
2
through the solution for 10 min followed by injecting
cies with potentially important pharmacological activities [24,25]. It
is a relatively unstable species, which rapidly decomposes forming the
transient hyponitrous acid that slowly produces nitrous oxide via dehy-
dration (reaction (4) [22,26]).
1
2 2
O
−
1
−1
(
ε
352=25,800 M
.3. EPR
EPR spectra were recorded using a JEOL X band JES-RE3X spectrom-
cm ) [33].
6
−1 −1
HNO þ HNO → H N O → N O þ H O
k ¼ 8 ꢂ 10 M
s
ð4Þ
2
2
2
2
2
4
2
The question arises whether the different nitroxides might be used
for HNO detection, quantification, and elucidation of the mechanism
underlying its formation. In the present study the rate constants of the
reactions of TEMPO, TEMPOL and 3-carbamoyl-PROXYL with HNO and
eter operating at 9.5 GHz with center field set at 3287 G, 100 kHz mod-
ulation frequency, 1 G modulation amplitude and 4–16 mW incident
microwave power. Samples were injected into a flexible capillary,
which was inserted into a quartz tube placed within the EPR spectrom-
eter cavity.
•
−
with N
2
O
2
have been determined. In addition, the ability of TEMPOL
and C-PTIO to oxidize HNO and their different reactivity toward NO
have been used to quantify HNO, which is known to be formed via the
oxidation of acetohydroxamic acid [7,27].
2.4. Radiolysis
2
. Materials and methods
Pulse radiolysis experiments were carried out using a 5-MeV Varian
7
715 linear accelerator (0.1–0.3 μs electron pulses, 200 mA current).
2
.1. Materials
A 200 W Xe lamp produced the analyzing light. Measurements were
done using 2 or 4 cm spectrosil cells with three light passes. Dosimetry
was performed with a N O-saturated solution containing 5 mM KSCN
2
Water for solution preparation was purified using a Milli-Q
system. All chemicals were of the highest available grade and
were used as received: acetohydroxamic acid (aceto-HX), TEMPOL,
TEMPO, 3-carbamoyl-PROXYL (3-CP), sodium trans-hyponitrite hy-
•
2
−
4
−1
−1
−1
using Gε((SCN)
)=5×10 radicals (100 eV)
M
cm
at 475 nm
34]. A 1 Cs source was used for steady-state radiolysis. Dosimetry was
37
[
II
.
performed with Fricke dosimeter (10 mM M Fe and 1 mM NaCl in 0.8 N
H
All experiments were carried out at room temperature.
Irradiation of aqueous solutions produces several species with differ-
ent yields as shown in Eq. (5). The numbers in parenthesis are G-values,
which represent the radiation yields in neutral water (in number of
molecules formed per 100 eV corresponding to 0.1036 μM Gy ) [35],
and are about 7% higher in the presence of high solute concentrations.
drate (Na
2 2
N O
2
xH
2
O), myoglobin from horse heart and Griess reagent
III
III
−1
−1
2
SO
4
) using G(Fe )=15.7 and ε(Fe )=2197 M cm
at 302 nm.
were purchased from Sigma-Aldrich. Catalase was purchased from
Boehringer Biochemicals. AS and C-PTIO were purchased from Cayman
Chemical Co. (San Diego, CA, USA). TEMPOL-H was prepared by bub-
bling HCl gas through ethanolic solution of TEMPOL followed by drying
[28]. Fresh solutions of TEMPOL-H were prepared immediately before
−
1
each experiment to minimize its oxidation to TEMPOL. Stock solution
of AS was prepared in 10 mM NaOH and its concentration was deter-
mined by the absorbance at 248 nm (ε=8300 M
−
1
−1
cm [29]). Stock
γ
−
•
•
þ
2−
−
H O → e ð2:6Þ; OHð2:7Þ; H ð0:6Þ; H O ð2:6Þ; H O ð0:72Þ
ð5Þ
solutions of N
2
O
2
(pK
a
(H
2
N
2
O
2
)=7.2, pK
a
(HN
2
O
2
)=11.5 [30]) were
2
aq
3
2
2
prepared in 0.1 M NaOH, and the concentration was determined by the
absorbance at 248 nm (ε(N
trations thus calculated correspond to 4 molecules of H
2
−
−1
−1
2
O
2
)=6550 M
cm ) [31]. The concen-
O per Na in
When solutions are saturated with N
2 2
O ([N O]=25 mM), the sol-
•
2
2 2 2
N O
vated electrons are converted into OH radicals (reaction (6)), and

Y. Samuni et al. / Journal of Inorganic Biochemistry 118 (2013) 155–161
157
•
2.0
under such conditions 90% of the primary radicals are OH and the
rest H radicals [35].
•
–
e
•
–
9
−1 −1
þ N O þ H O→ OH þ N þ OH
k ¼ 9:1 ꢂ 10 M
s
ð6Þ
1.5
aq
2
2
2
6
1
0
0
.0
.5
.0
3
. Results and discussion
.1. HNO reaction with nitroxides
The reactions of TEMPOL, TEMPO and 3-CP with HNO have been
3
TEMPOL
TEMPO
3-CP
studied using AS. The EPR-signal of 100 μM nitroxide progressively
disappeared upon exposure to excess of AS in aerated solutions
containing 40 mM phosphate buffer at pH 7.0. The initial signal was
fully restored upon the addition of 1 mM ferricyanide indicating
that RNO is reduced by HNO to its respective EPR-silent RNO-H.
The rate constants of HNO reactions with RNO have been deter-
0
10
20
30
40
[nitroxide]/[MbFeIII]
II
Fig. 2. Nitroxide effect on the initial rate of MbFe NO formation analyzed according to
Eq. (8). The formation of MbFe NO was monitored at 425 nm upon mixing 42–44 μM AS
with 10 or 22 μM MbFe in aerated solutions containing 10 mM PB at pH 7.0 and 23 °C.
II
III
mined using competition kinetics against MbFe . In the presence of
AS, MbFe undergoes reductive nitrosylation, producing a ferrous-
III
III
nitrosyl complex, which is rather stable (reaction (7)) [36–38].
low in view of the relatively high driving force for this reaction calculat-
MbFe þ HNO → MbFe NO þ Hþ
III
II
ð7Þ
ed using E (NO/HNO)=−0.55 V [22,23] and E (RNO/RNO-H)≈0.2 V
7
7
for TEMPO and TEMPOL [13,16], ≈0.1 V for 3-CP [13] and ≈0.27 V
The addition of 40–50 μM AS to aerated solutions containing
0–30 μM MbFe at pH 7 yielded, within the accuracy of the mea-
surements, the same initial rate of MbFe NO formation (V
was monitored at 425 nm. This observation implies that under such
experimental conditions the rates of reaction (4) and that of HNO
for C-PTIO [17] (Table 1).
III
1
Reaction (1) can proceed via a single concerted step, i.e., hydrogen
atom transfer (HAT), or via stepwise processes involving initial electron
transfer (ET) followed by proton transfer (PT), or PT followed by ET as
shown in Scheme 2. It has been demonstrated that the TEMPO/TEMPOH
and related redox couples are particularly valuable for proton-coupled
electron transfer studies because of the their low O\H bond strengths,
and their strong thermochemical bias toward HAT reactions [39,40].
II
o
), which
3
−1 −1
with O
2
(k≈3×10
M
s
[4]) are negligible as compared to the
II
rate of reaction (7). Therefore, the initial rate of MbFe NO formation
V) was followed upon mixing 44 μM AS with 10 or 22 μM MbFe
and varying [RNO]. Fig. 1 demonstrates typical results obtained in
the case of TEMPOL. The effect [RNO] on the initial rate of MbFe NO
III
(
o
−
Similar arguments also hold for HNO (pK =11.4, E (NO/NO )=
a
II
−0.8 V [22,23]) whose bond dissociation free energy (BDFE) in water
−
1
formation follows relationship 8.
is estimated according to Eq. (9) to be≈55 kcal mol , which is signif-
−1
icantly lower than that of RNO-H, e.g., ≈71 kcal mol
[40].
for TEMPO-H
III
1
=V ¼ 1=V þ ðk =k V Þð½RNOꢃ=½MbFe ꢃÞ
ð8Þ
o
1
7
o
III
o
In Fig. 2 plots of V
/k =0.17± 0.02 for TEMPO and TEMPOL and 0.054± 0.004 for
1
-CP. Using k =(1.4± 0.2)×
o
/V−1 vs. [RNO]/[MbFe ] are given yielding
BDFEðX−HÞwater ¼ 1:37pK þ 23:06E þ 57:6
ð9Þ
a
k
3
1
1
7
5
−1 −1
7
=8×10
M
s
[4], one calculates k
for TEMPOL and TEMPO and (4.3± 0.4)×10
for 3-CP. The former value is ca. twice higher than the values reported
in the literature and close to those previously determined for PTIO
and C-PTIO (Table 1). The reactivity of HNO toward RNO is surprisingly
The rate constant of HAT reaction can be predicted using the Marcus
cross relation, which was originally developed for electron transfer reac-
tion, but has been demonstrated to provide a valuable new framework
for understanding and predicting HAT reactivity [41,42]. Accordingly,
5
−1 −1
4
−1 −1
0
M
s
M
s
k
1
is calculated using Eqs. (10) and (11) where kHNO/NO and kRNO-H/RNO
are the HNO/NO and RNO-H/RNO self-exchange rate constants.
0
0
0
0
0
.72
.68
.64
.60
.56
ꢀ
ꢁ
1=2
k1 ¼ kHNO=NO
k
K f
ð10Þ
ð11Þ
RNO−H=RNO
1
n
ꢀ
ꢁo
2
22
ln f ¼ ðlnK Þ = 4ln kHNO=NOk
=10
RNO−H=RNO
1
Table 1
Driving force and rate constants for the reaction of HNO with nitroxides at pH 7.
Nitroxide
ΔG
1
°
k
1
, M−1s−1
Ref.
−
(
kcal mol 1)a
5
4
5
TEMPO
−17.3
−17.3
(1.4± 0.2)×10
(6.3± 0.8)×10
(1.4± 0.2)×10
This work
[5]
This work
[4]
This work
[6]
0
30
60
90
120
150
TEMPOL
Time (s)
4
8
×10
4
5
3
-CP
C-PTIO
−15.0
−18.9
(4.3± 0.4)×10
(1.4± 0.2)×10
II
Fig. 1. TEMPOL effect on the initial rate of MbFe NO formation. The formation of
MbFe NO was monitored at 425 nm upon mixing 44 μM AS with 10 μM MbFe in
the absence and presence of 21, 42 and 83 μM TEMPOL in aerated solutions containing
II
III
a
Calculated using E
7
7
(NO/HNO)=−0.55 V [22,23] and E (RNO/RNO-H)=0.2 V for
1
0 mM PB at pH 7.0 and 23 °C.
TEMPO and TEMPOL [13,16], 0.1 V for 3-CP [13] and 0.27 V for C-PTIO [17].

158
Y. Samuni et al. / Journal of Inorganic Biochemistry 118 (2013) 155–161
2
1
1
000
500
000
TEMPO
TEMPOL
3-CP
Scheme 2. Concerted vs. stepwise transfer of H++e− demonstrated for reaction (1).
500
The TEMPO-H/TEMPO self-exchange rate constant has been esti-
mated from pseudo-self-exchange reactions between similar nitroxides
0
.0
0.3
0.6
0.9
1.2
−
1
−1
[nitroxide] (mM)
to be 4.7± 1.0 M
s
in MeCN [39]. We have estimated a value of
in water using the Litwinienko and Ingold's kinetic
solvent effect model, i.e., Eq. (12) [43], and the Abraham H-bonding
−
1
−1
7
.4± 1.6 M
s
Fig. 3. Observed first order rate constant for the decay of the hyponitrite radical in the
presence of various concentrations of TEMPO, TEMPOL and 3-CP. The decay of the
hyponitrite radical was monitored at 290 nm upon pulse irradiation (1.76 Gy/pulse)
H
H
H
parameters α
.38 [44]. The origin of this very slow self-exchange rate constant has
been discussed elsewhere [39].
2 2 2 2
(TEMPO-H)=0.39, β (MeCN)=0.44 and β (H O)=
0
of N
2
O-saturated solutions containing 4 mM sodium trans-hyponitrite and 40 mM PB
(pH 6.8).
logðksolv1Þ − logðksolv2
Þ
ð12Þ
•
−
2 2
N O shows surprisingly low reactivity toward RNO given that the
h
i
H
H
H
o
+
¼
−8:3α2 ðTEMPO
ꢀ
HÞ β ðsolv1Þ−β2 ðsolv2Þ
midpoint potential at pH 6.8 is≈1.25 V [11] and E (RN =O/RNO)=
.74, 0.81 and 0.87 V for TEMPO, TEMPOL and 3-CP, respectively
[12,14–16], i.e., ΔG14≈−(9–12)kcal mol
2
0
o
−1
Using kTEMPO-H/TEMPO =7.4 M−1 s−1 and K
12
.
1
=5.4×10 , the calcu-
5
−1 −1
lated k
HNO/NO≈0.03 M
latter value can vary within 1–2 orders of magnitude [41]. The experi-
mental k and K values for TEMPO and TEMPOL are the same implying
that the kRNO-H/RNO values for these nitroxides are similar. Assuming that
1
approaches the experimental value (1.4×10
M
s
) when
−
1
−1
•−
2
2−
þ
k
s
. Within the limitations of this approach, the
N O þ RNO → N O þ RN ¼O
ð14Þ
2
2
2
•
−
•−
1
1
Low reactivity of N
2
O
towards O
and O
at pH 9.2 has previ-
2
2
2
ously been reported for these strongly driven reactions with corre-
this is also the case for 3-CP, one calculates k
1
=2×104
M
−1
s
−1
, which
sponding rate constants below 1×106 and 5×107
M
−1
s
−1
[11]. It
is ca. twice lower than the experimental value. It is concluded that the
slowness of reaction (1) can be attributed to the low self-exchange
rate constants of HNO/NO and RNO-H/RNO.
has been assumed that there are large nuclear reorganization barriers
for both reduction and oxidation of N O , and that these processes
2 2
proceed via an inner-sphere electron transfer mechanism [11].
Oxidation of HNO by piperidine and pyrrolidine nitroxides yields
NO, which does not react with these nitroxides but rather with HNO
forming the hyponitrite radical (reaction (2)). This radical can either
•−
3
.2. The reaction of hyponitrite radical with nitroxides and TEMPOL-H
Hyponitrite radicals were generated in situ upon pulse-irradiation
react with RNO/RNO-H or add to NO forming eventually N
nitrite (reactions (15), (16)) [10].
2
O and
2
of N O-saturated solutions containing 4 mM sodium trans-hyponitrite
and 40 mM phosphate buffer (pH 6.8).
•
2
−
−
9
−1 −1
N O þ NO → N O
k15 ¼ 5:4 ꢂ 10
M
s
ð15Þ
ð16Þ
•
−
•
2
•−
2
2
3
3
OH þ H N O =HN O → HN O =N O þ H O
ð13Þ
2
2
2
2
2
2
2
2
−
−
k16 ¼ 300 s−1
N O þ NO → N O þ NO
The apparent rate constant of the hyponitrite radicals formation
3
3
2
2
at pH 6.8 is about 1×109
M
−1
s
−1
•
2 2
[11,45]. The HN O radical is
Since k14 =(5.9 – 24)×105 M−1 s−1 is about 4-orders of magni-
tude lower than k15, the contribution of reaction (14) is insignificant
and net reaction (17) is obtained for RNO reduction by HNO.
weakly acidic (pK
ical predominates. The decay of N
a
=5.5 [11,45]) and at pH 6.8 the deprotonated rad-
•−
2
O
2
was monitored at 290 nm
−
1
−1
(
ε=5900 M
netics (2kapp =4.2×10
values [11,45]. The decay of N
order kinetics in the presence of the nitroxides using a low pulse in-
tensity (1.76 Gy, [OH]
cm
[45]), and was found to obey second-order ki-
8
−1 −1
M
s
) in agreement with the literature
•
−
2RNO þ 3HNO → 2RNOꢀH þ N O þ NO þ Hþ
−
2
O
2
turned from second- to first-
ð17Þ
2
2
o
≈1 μM) to minimize the contribution of the
•
3
.3. Oxidation of aceto-HX by OH in the presence of TEMPOL or C-PTIO
second-order decomposition. The observed first-order rate constants
increased upon increasing the concentration of the nitroxides (Fig. 3),
and the bimolecular rate constants were derived from the slopes of
•
2
OH radical was generated upon irradiation of N O-saturated solu-
6
−1 −1
a
tions containing 4 mM aceto-HX (pK =9.0 [48]), 100 μM nitroxide,
the lines in Fig. 3 to be (2.4± 0.2)×10
M
s
for TEMPO, (9.8±
−
1
5
−1 −1
5
−1 −1
55 U mL
catalase and 10 mM phosphate buffer at pH 8.3. Under
0
3
.2)×10
M
s
for TEMPOL and (5.9± 0.5)×10
M
s
for
•
•−
such conditions OH reacts exclusively with the hydroxamate moiety
forming the transient nitroxide radical (reaction (18)) [7].
-CP. The yield of N
2
O
2
decreased upon increasing the concentration
−
of the nitroxides due to the competition between H
2
N
2
O
2
/HN
M
2
O
s
2
•
•
9
−1 −1
and the nitroxides for OH (k( OH+nitroxide)=(3–5)×10
•
−
•
•−
OH þ CH CðOÞNHOH=CH CðOÞNHO → CH CðOÞNHO =CH CðOÞNO
[
46,47]), but we assume that the resulting alkyl radicals and/or
3
3
3
3
+
•−
−
2 2
RN =O have no effect on the decay of N O . The rate constant
of TEMPOL-H reaction with N
1
þ H O=OH
ð18Þ
2
•
−
2
O
2
was estimated to be k≈3×
4
−1 −1
•−
0
M
s
based on the decrease of the half-life of the N
2
O
2
by
Catalase was added to the reaction mixture to remove the
radiolytically formed H , which is capable of oxidizing RNO-H.
about 50% in the presence of 4 mM TEMPOL-H.
2 2
O

Y. Samuni et al. / Journal of Inorganic Biochemistry 118 (2013) 155–161
159
1
00
no additive
5
4
3
2
1
0
8
6
4
2
0
0
0
0
0
C-PTIO
TEMPOL
aceto-HX
120
0
10
20
30
0
30
60
90
150
Irradiation Time (min)
Incubation Time (min)
•
Fig. 4. TEMPOL and C-PTIO depletion induced upon aceto-HX oxidation by OH. Solutions
2 2 2 2
Fig. 5. Effects of TEMPOL and C-PTIO on H O depletion. Depletion of 5 mM H O in
O containing 100 μM nitroxide, 55 U mL− catalase, 4 mM aceto-HX
1
III
saturated with N
and 10 mM phosphate buffer (pH 8.3) were irradiated at a dose rate of 7.8 Gy min
O) TEMPOL; (●) C-PTIO. The EPR signal intensity was monitored using modulation
G, power 8 mW, field center 3287 G and TC 0.1 s.
2
10 mM phosphate buffer (pH 7.0) containing 25 μM MbFe : (O) no additive; (●) 4 mM
aceto-HX; (■) 4 mM aceto-HX and 100 μM TEMPOL; (□) 4 mM aceto-HX and 100 μM
C-PTIO.
−
1
:
(
1
III
3
.4. Oxidation of aceto-HX by MbFe /H
O
2 2
in the presence of TEMPOL or
The EPR-signal of TEMPOL and C-PTIO decreased linearly with the
C-PTIO
−
1
radiation dose with a depletion rate of 1.4± 0.1 μM min
(Fig. 4).
MbFeIII in the presence of H O2 demonstrates a modest catalase-
like activity (reactions (19)–(21)), which is stimulated in the pres-
ence of aceto-HX [27] or nitroxides [50–52].
The conversion of C-PTIO to C-PTI was not observed, which can be
attributed to the accumulation of C-PTIO-H and/or C-PTI-H. The addi-
tion of 1 mM ferricyanide restored most of the EPR signal of TEMPOL
and C-PTIO, though not of C-PTI, indicating that C-PTI-H was not accu-
mulated in this system. The rate of nitrite formation was significantly
2
III
•
IV
MbFe þ H O → MbFe ¼O þ H O
ð19Þ
ð20Þ
ð21Þ
2
2
2
−
1
low, 0.06± 0.02 μM min , and did not increase upon subsequent expo-
sure of the anoxic irradiated samples to oxygen. The rate of RNO reduc-
•
MbFe ¼O þ H O → MbFe ¼O þ O þ 2Hþ
IV
IV
•−
2
2
2
•
tion by HNO is an upper limit value since the radiolytically produced H
−
1
IV
III
•−
radical (0.47 μM min ) can also reduce RNO [46]. Thus, the lower re-
duction rate of RNO is 0.9± 0.1 μM min , and since the rate of OH pro-
duction was 4.5 μM min , the yields of HNO vary between 20 and 31%
depending on the competition between 4 mM aceto-HX and 100 μM
MbFe ¼O þ H O → MbFe þ O2 þ H O
2
2
2
−
1
•
−
1
The addition of 100 μM TEMPOL or C-PTIO to solutions containing
III
4 mM aceto-HX, 25 μM MbFe and 5 mM H O2 facilitates H O2 de-
2
2
•
RNO for H radical.
pletion (Fig. 5) and nitrite accumulation (Fig. 6). Upon the addition
III
These results demonstrate that the yield of HNO is in agreement
with that previously determined by measuring the accumulation of
of H O2 to solutions containing MbFe , aceto-HX and nitroxide, the
2
III
spectrum of MbFe is converted within less than 30 s into that of
IV
III
2
N O at pH 7.0 (ca. 25% [27]), and that NO formed via the oxidation of
MbFe =O implying that the steady-state concentration of MbFe is
extremely low as evident from the absence of its characteristic absor-
bance at 408 nm. About 30% of the EPR signal of TEMPOL was lost
during the consumption of most H O . The EPR-signal could not be
HNO by TEMPOL or C-PTIO, is efficiently trapped by species other than
HNO or C-PTIO. The spin density on the transient nitroxide radical
derived upon one-electron oxidation of aceto-HX is evenly distributed
over the O\C\N\O, and the potential tautomeric radicals are shown
in Scheme 3. We have previously demonstrated that the transient
2
2
restored upon the addition of ferricyanide demonstrating the conver-
sion of TEMPOL to diamagnetic species other than TEMPOL-H. In the
case of C-PTIO, about 50% of its EPR signal decayed while only about
•
•−
3 3
radicals CH C(O)NHO /CH C(O)NO decompose bimolecularly [7,27].
The recombination of such tautomeric radicals can presumably produce
various adducts where the O\O ones are expected to be unstable. We
have assumed that these adducts decompose via heterolysis of the
10% was converted into C-PTI during the consumption of most H O2
(Fig. 7). The EPR-signals of C-PTIO and C-PTI were unaffected by the
addition of ferricyanide indicating that the respective hydroxyl-
amines were not accumulated. These results demonstrate that
2
O\O bond yielding CH
HNO via hydrolysis. Since the yield of HNO derived upon TEMPOL/
C-PTIO reduction is similar to that calculated from the yield of N
3 3
C(O)NHOH and CH C(O)N=O, which forms
TEMPOL and C-PTIO act catalytically in facilitating H O2 decomposi-
2
2
O
tion and nitrite formation. The 30–50% loss of the nitroxides during
in the absence of these nitroxides, it is suggested that NO is effi-
ciently trapped by the carbon-centered radical [49] and possibly by
the nitrogen-centered one (see Scheme 2).
the complete consumption of 5 mM H O can be attributed to their abil-
2
2
•
IV
•
ity to react with carbon-centered radical, e.g., MbFe =O, CH C(O)NHO
3
(Scheme 2), forming stable diamagnetic adducts [53,54].
3
Scheme 3. Tautomeric radicals derived from one-electron oxidation of CH C(O)NHOH.

160
Y. Samuni et al. / Journal of Inorganic Biochemistry 118 (2013) 155–161
1
1
1
1
600
400
200
000
2 2
catalytically facilitate the decomposition of H O and accumulation of ni-
trite. In this system the conversion of C-PTIO into C-PTI is only a minor
side reaction, which does not provide any mechanistic insight.
none
C-PTIO
TEMPOL
Abbreviations
aceto-HX acetohydroxamic acid
8
6
4
2
00
00
00
00
0
AS
Angeli's salt
BDFE
bond dissociation free energy
3-carbamoyl-PROXYL
3
-CP
C-PTI
2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl
C-PTIO 2-(4-carboxyphenyl)-4,4,5,5,-tetramethyl-imidazoline-1-
oxyl-3-oxide
0
10
20
30
40
50
60
70
ET
electron transfer
Incubation time (min)
HAT
HX
hydrogen atom transfer
hydroxamic acid
Fig. 6. Effects of TEMPOL and C-PTIO on nitrite accumulation. Solutions contained
and 4 mM aceto-HX in 10 phosphate buffer at pH 7.0
Δ) with 100 μM TEMPOL (O) or 100 μM C-PTIO (●).
IV
MbFe = O ferryl myoglobin
MbFe
PT
III
2
(
5 μM MbFe , 5 mM H
2
O
2
III
metmyoglobin (MetMb)
proton transfer
TEMPO 2,2,6,6-tetramethyl piperidine-1-oxyl
TEMPOL 4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl
TEMPOL-H TEMPOL-hydroxylamine
Accumulation of nitrite and the conversion of C-PTIO to C-PTI have
been previously demonstrated during the oxidation of hydroxyurea,
SAHA and Trichostatin A by heme proteins and H , which led to the
conclusion that these hydroxamic acids release NO upon oxidation
55–57]. However, since C-PTIO catalytically facilitates H decompo-
O
2 2
RNO
nitroxide
RNO-H hydroxylamine
[
2 2
O
sition and nitrite accumulation, the minor conversion of C-PTIO to
C-PTI demonstrates the formation of NO, but does not provide any
mechanistic insight since NO can be produced via HNO oxidation by
Acknowledgments
IV
C-PTIO and/or MbFe =O. The very low yield of C-PTI implies that
This work has been supported by the Israel Science Foundation
IV
MbFe =O competes efficiently with C-PTIO for NO, and that reac-
(
Grant No. 1477) and by the Queens College Research Enhancement
tion (22) is the main source of nitrite in this system.
Funds and PSC-CUNY Research Awards.
MbFe ¼O þ NO → MbFe þ NO2− k22 ¼ 1:79 ꢂ 10 M
IV
III
7
−1
s
−1[58]ð22Þ
References
[
[
[
[
1] C.S. Lai, L.E. Hopwood, H.M. Swartz, Exp. Cell Res. 130 (1980) 437–442.
2] P.D.J. Morse, H.M. Swartz, Magn. Reson. Med. 2 (1985) 114–127.
3] H.M. Swartz, Free Radic. Res. Commun. 9 (1990) 399–405.
4] K.M. Miranda, N. Paolocci, T. Katori, D.D. Thomas, E. Ford, M.D. Bartberger, M.G.
Espey, D.A. Kass, M. Feelisch, J.M. Fukuto, D.A. Wink, Proc. Natl. Acad. Sci. U. S. A.
4
. Conclusions
•
−
The reactivity of HNO and N
2
O
2
toward cyclic nitroxides is sur-
100 (2003) 9196–9201.
prisingly low given the considerable driving force for these reactions.
Cyclic nitroxides are reduced to their respective hydroxylamines by
HNO. The rate constants for these HAT reactions can be predicted
using the Marcus cross relation assuming that the self-exchange rate con-
stants of HNO/NO and RNO-H/RNO are significantly low. Cyclic nitroxides
cannot be used as quantitative HNO scavengers when hydroxamic acids
[
5] M.I. Jackson, T.H. Han, L. Serbulea, A. Dutton, E. Ford, K.M. Miranda, K.N. Houk,
D.A. Wink, J.M. Fukuto, Free Radic. Biol. Med. 47 (2009) 1130–1139.
[6] U. Samuni, Y. Samuni, S. Goldstein, J. Am. Chem. Soc. 132 (2010) 8428–8432.
[7] S. Goldstein, A. Samuni, J. Phys. Chem. A 115 (2011) 3022–3028.
[
8] M. Gratzel, S. Taniguchi, A. Henglein, Ber. Bunsenges. Phys. Chem. 74 (1970)
003–1010.
9] W.A. Seddon, J.W. Fletcher, F.C. Sopchyshyn, Can. J. Chem. 51 (1973) 1123–1130.
1
[
are oxidized by the heme proteins and H
2
O
2
reaction system since they
[10] S.V. Lymar, V. Shafirovich, G.A. Poskrebyshev, Inorg. Chem. 44 (2005) 5212–5221.
[11] G.A. Poskrebyshev, V. Shafirovich, S.V. Lymar, J. Am. Chem. Soc. 126 (2004) 891–899.
[12] J.R. Fish, S.G. Swarts, M.D. Sevilla, T. Malinski, J. Phys. Chem. 92 (1988) 3745–3751.
[13] Y. Kato, Y. Shimizu, L. Yijing, K. Unoura, H. Utsumi, T. Ogata, Electrochim. Acta 40
(1995) 2799–2802.
[
[
[
14] V.G. Granik, N.A. Grigor'ev, Russ. Chem. Bull. 51 (2002) 1375–1422.
15] S. Goldstein, A. Samuni, A. Russo, J. Am. Chem. Soc. 125 (2003) 8364–8370.
16] A. Israeli, M. Patt, M. Oron, A. Samuni, R. Kohen, S. Goldstein, Free Radic. Biol. Med.
38 (2005) 317–324.
17] S. Goldstein, A. Russo, A. Samuni, J. Biol. Chem. 278 (2003) 50949–50955.
18] T. Akaike, M. Yoshida, Y. Miyamoto, K. Sato, M. Kohno, K. Sasamoto, K. Miyazaki, S.
Ueda, H. Maeda, Biochemistry 32 (1993) 827–832.
t = 0
[
[
[
19] N. Hogg, R.J. Singh, J. Joseph, F. Neese, B. Kalyanaraman, Free Radic. Res. 22 (1995)
4
7–56.
20] J. Joseph, B. Kalyanaraman, J.S. Hyde, Biochem. Biophys. Res. Commun. 192 (1993)
26–934.
[
9
[
[
[
21] T. Akaike, H. Maeda, Methods Enzymol. 268 (1996) 211–221.
22] V. Shafirovich, S.V. Lymar, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 7340–7345.
23] M.D. Bartberger, W. Liu, E. Ford, K.M. Miranda, C. Switzer, J.M. Fukuto, P.J. Farmer,
D.A. Wink, K.N. Houk, Proc. Natl. Acad. Sci. U. S. A. 99 (2002) 10958–10963.
24] N. Paolocci, M.I. Jackson, B.E. Lopez, K. Miranda, C.G. Tocchetti, D.A. Wink, A.J.
Hobbs, J.M. Fukuto, Pharmacol. Therapeut. 113 (2007) 442–458.
t = 50 min
[
[
25] C.H. Switzer, W. Flores-Santana, D. Mancardi, S. Donzelli, D. Basudhar, L.A. Ridnour, K.M.
Miranda, J.M. Fukuto, N. Paolocci, D.A. Wink, Biochim. Biophys. Acta 1787 (2009)
Fig. 7. Change of C-PTIO EPR spectrum upon exposure of aceto-HX to oxidative stress.
8
35–840.
III
C-PTIO at 100 μM C-PTIO, 4 mM aceto-HX, 25 μM MbFe and 5 mM H
phosphate buffer at pH 7.0 were incubated. Top: the five-line spectrum of C-PTIO before
the addition of H ; Bottom: the decay of C-PTIO and the appearance of C-PTI after
0 min of incubation. The EPR spectra were monitored using modulation 1 G, Power
mW, field center 3287 G, gain 160 and TC 0.03 s.
2 2
O in 10 mM
[
[
26] M.C. Lin, Y.S. He, C.F. Melius, Int. J. Chem. Kinet. 24 (1992) 489–516.
27] Y. Samuni, U. Samuni, S. Goldstein, Biochim. Biophys. Acta 1820 (2012) 1560–1566.
[28] R.I. Zhdanov, P.G. Komarov, Free Radic. Res. 9 (1990) 367–377.
[29] C.C. Addison, G.A. Gamlen, R. Thompson, J. Chem. Soc. (1952) 338–345.
[30] F.T. Bonner, M.N. Hughes, Comments Inorg. Chem. 7 (1988) 215–234.
2
O
2
5
4

Y. Samuni et al. / Journal of Inorganic Biochemistry 118 (2013) 155–161
161
[
[
31] C.N. Polydoropoulos, S.D. Voliotis, Anal. Chim. Acta 40 (1968) 170–172.
32] E. Antonini, M. Brunori, Hemoglobin and Myoglobin in Their Reactions with
Ligands, North-Holland, Amsterdam, 1971.
33] N.V. Klassen, D. Marchington, H.C.E. McGowan, Anal. Chem. 66 (1994) 2921–2925.
34] G.V. Buxton, C.R. Stuart, J. Chem. Soc. Faraday Trans. 91 (1995) 279–281.
35] G.V. Buxton, Q.G. Mulazzani, A.B. Ross, J. Phys. Chem. Ref. Data 25 (1995) 1055–1349.
36] D.A. Bazylinski, T.C. Hollocher, J. Am. Chem. Soc. 107 (1985) 7982–7986.
37] M.P. Doyle, S.N. Mahapatro, R.D. Broene, J.K. Guy, J. Am. Chem. Soc. 110 (1988)
[46] K.D. Asmus, S. Nigam, R.L. Willson, Int. J. Radiat. Biol. Relat. Stud. Phys. Chem.
Med. 29 (1976) 211–219.
[47] A. Samuni, S. Goldstein, A. Russo, J.B. Mitchell, M.C. Krishna, P. Neta, J. Am. Chem.
Soc. 124 (2002) 8719–8724.
[48] B. Monzyk, A.L. Crumbliss, J. Org. Chem. 45 (1980) 4670–4675.
[49] G. Czapski, J. Holcman, B.H.J. Bielski, J. Am. Chem. Soc. 116 (1994) 11465–11469.
[50] R.J. Mehlhorn, C.E. Swanson, Free Radic. Res. Commun. 17 (1992) 157–175.
[51] M.C. Krishna, A. Samuni, J. Taira, S. Goldstein, J.B. Mitchell, A. Russo, J. Biol. Chem.
271 (1996) 26018–26025.
[
[
[
[
[
593–599.
[
38] K.M. Miranda, R.W. Nims, D.D. Thomasa, M.G. Espey, D. Citrin, M.D. Bartberger, N.
Paolocci, J.M. Fukuto, M. Feelisch, D.A. Wink, J. Inorg. Biochem. 93 (2003) 52–60.
39] A. Wu, E.A. Mader, A. Datta, D.A. Hrovat, W.T. Borden, J.M. Mayer, J. Am. Chem.
Soc. 131 (2009) 11985–11997.
40] J.J. Warren, T.A. Tronic, J.M. Mayer, Chem. Rev. 110 (2010) 6961–7001.
41] J.M. Mayer, Science 294 (2001) 2524–2526.
[52] O.M. Lardinois, D.A. Maltby, K.F. Medzihradszky, P.R.O. de Montellano, K.B. Tomer,
R.P. Mason, L.J. Deterding, Chem. Res. Toxicol. 22 (2009) 1034–1049.
[53] V.W. Bowery, K.U. Ingold, J. Am. Chem. Soc. 114 (1992) 4992–4996.
[54] M.A. Lam, D.I. Pattison, S.E. Bottle, D.J. Keddie, M.J. Davies, Chem. Res. Toxicol. 21
(2008) 2111–2119.
[55] R. Pacelli, J. Taira, J.A. Cook, D.A. Wink, M.C. Krishna, Lancet 347 (1996) 900.
[56] J.M. Huang, E.M. Sommers, D.B. Kim-Shapiro, S.B. King, J. Am. Chem. Soc. 124 (2002)
3473–3480.
[
[
[
[
[
[
[
42] J.M. Mayer, Acc. Chem. Res. 44 (2011) 36–46.
43] G. Litwinienko, K.U. Ingold, Acc. Chem. Res. 40 (2007) 222–230.
44] J.J. Warren, J.M. Mayer, Proc. Natl. Acad. Sci. U. S. A. 107 (2010) 5282–5287.
45] G.A. Poskrebyshev, V. Shafirovich, S.V. Lymar, J. Phys. Chem. A 112 (2008)
[57] Y. Samuni, W. Flores-Santana, M.C. Krishna, J.B. Mitchell, D. Wink, Free Radic. Biol.
Med. 47 (2009) 419–423.
8295–8302.
[58] S. Herold, F.J.K. Rehmann, J. Biol. Inorg. Chem. 6 (2001) 543–555.