Evaluation of Activation Volumes for the Conversion of Peroxynitrous to Nitric Acid

Article abstract of DOI:10.1021/jp0362988

Peroxynitrous acid, an inorganic toxin of biological importance, acts both as an oxidizing and a nitrating agent during its conversion to nitric acid. In discussions of the mechanism of this conversion, activation volumes have been invoked to distinguish between possible mechanisms, viz., homolysis of the O-O bond versus rotation via the N-O bond of peroxynitrous acid. A reinvestigation of the activation volume for the conversion of peroxynitrous acid to nitric acid by high-pressure stopped-flow spectrophotometry yielded an average value of 6.9 ± 0.9 cm³ mol^-1 at 25 °C. Activation volumes currently cited in the literature for this process range from 6 to 10 cm³ mol^-1 in the temperature range 18-25 °C. Such moderately positive values do not support a definite conclusion regarding the mechanism of the conversion.

Full text of DOI:10.1021/jp0362988

© Copyright 2003 by the American Chemical Society
VOLUME 107, NUMBER 51, DECEMBER 25, 2003
LETTERS
Evaluation of Activation Volumes for the Conversion of Peroxynitrous to Nitric Acid
Reinhard Kissner,† Chris Thomas,† Mohamed S. A. Hamsa,‡ Rudi van Eldik,*,‡ and
Willem H. Koppenol*,†
Institute of Inorganic Chemistry, Swiss Federal Institute of Technology, CH-8093 Zu¨rich, Switzerland, and
Institute of Inorganic Chemistry, UniVersity of Erlangen-Nu¨rnberg, D-91058 Erlangen, Germany
ReceiVed: August 5, 2003; In Final Form: NoVember 10, 2003
Peroxynitrous acid, an inorganic toxin of biological importance, acts both as an oxidizing and a nitrating
agent during its conversion to nitric acid. In discussions of the mechanism of this conversion, activation
volumes have been invoked to distinguish between possible mechanisms, viz., homolysis of the O-O bond
versus rotation via the N-O bond of peroxynitrous acid. A reinvestigation of the activation volume for the
conversion of peroxynitrous acid to nitric acid by high-pressure stopped-flow spectrophotometry yielded an
average value of 6.9 ( 0.9 cm3 mol-1 at 25 °C. Activation volumes currently cited in the literature for this
process range from 6 to 10 cm3 mol-1 in the temperature range 18-25 °C. Such moderately positive values
do not support a definite conclusion regarding the mechanism of the conversion.
Peroxynitrite1 is formed in vivo from the diffusion-controlled
reaction of superoxide with nitrogen monoxide, k ) (1.6 ( 0.3)
× 1010 M-1 s-1.2 This reaction is biologically relevant especially
near activated macrophages, which produce micromolar con-
centrations of nitrogen monoxide. In contrast, inside cells where
nitrogen monoxide is found at lower signal-level concentrations
(nM), the presence of ca. 10 µM superoxide dismutase prevents
the reaction with superoxide, and peroxynitrite formation is
unlikely. While at physiological pH a significant part of the
peroxynitrite combines with carbon dioxide3 to form an adduct
that may be oxidizing itself and decays mostly to carbon dioxide
and nitrate,4 at lower pH and inside membranes, the decay of
peroxynitrous acid may become physiologically relevant. During
this decay, a strong oxidant is formed, the nature of which is a
matter of debate. The current mechanistic viewpoints are that
the conversion of peroxynitrous to nitric acid can proceed by
way of homolysis of the O-O bond to produce free nitrogen
dioxide and hydroxyl radicals to an extent of 10-40%,5-8 or
via a rotation along the N-O bond followed by an intramo-
lecular HO transfer.9-11 Activation volumes have been invoked
by disputing research groups in attempts to differentiate between
these two possible mechanisms. Initially, small positive values
that favor the intramolecular HO-transfer mechanism were
found, but in later studies, authors reported significantly larger
values that were interpreted in terms of a homolysis mechanism.
In the meantime, the research groups involved have repeated
their measurements; herein we report our evaluation of the
existing data.
Tetramethylammonium peroxynitrite was synthesized and
purified according to Bohle et al.12 Starting materials were
synthetic-grade potassium superoxide from Aldrich, tetramethyl-
ammonium hydroxide from Fluka, and 99.9% nitrogen mon-
oxide from Linde. A solution of peroxynitrite at ca. 1 mM was
prepared in freshly prepared 10 mM analytical-grade sodium
hydroxide (Siegfrid) and frozen at -80 °C in small quantities.
Immediately prior to an experiment, the frozen peroxynitrite
pellets were dissolved in freshly prepared 10 mM sodium
hydroxide cooled in an ice bath in quantities suitable to obtain
* Corresponding authors. E-mail: R.v.E., vaneldik@chemie.uni-erlangen.
de; W.H.K., koppenol@inorg.chem.ethz.ch.
† HCI H211, ETH Ho¨nggerberg, Swiss Federal Institute of Technology.
‡ University of Erlangen-Nu¨rnberg.
10.1021/jp0362988 CCC: $25.00 © 2003 American Chemical Society
Published on Web 11/27/2003

11262 J. Phys. Chem. A, Vol. 107, No. 51, 2003
Letters
ref
-
TABLE 1: Activation Volumes for the Reaction ONOOH f NO3 + H+
activation vol/
cm3 mol-1
conditions
technique
9.6 ( 1.0
10.7 ( 1.9
10.0 ( 0.3
10.5 ( 1.1
6.0 ( 0.7
9.7 ( 1.4
6.2 ( 1.1
7.6 ( 1.3
7.8 ( 1.1
6.0 ( 0.5
6.6 ( 1.0
4.8 ( 0.2
pH 4.1, 0.15 M HCO2-, 3 mM NO2-, P ) 0.1 and 150 MPa, ambient T
pH 4.1, 0.15 M HCO2-, 3 mM NO2-, P f 150 MPa, 18 °C
pH 4.1, 3 mM NO3-, 4 mM H2PO4-, P f 150 MPa, 19 °C
pH 5.6, 3 mM NO3-, 4 mM H2PO4-, P f 150 MPa, 18-19 °C
pH 4.5, 0.15 M H2PO4-, 53 µM NO2-, P f 100 MPa, 19.5 °C
pH 4.5, 0.15 M H2PO4-, 0.5 f 15 mM NO2-, P f ? MPa, 20 °C
pH 3.6, 5 mM H2PO4-, 50 f 150 MPa, 25 °Ca
pulse radiolysis
pulse radiolysis
pulse radiolysis
pulse radiolysis
stopped-flow
stopped-flow
stopped-flow
stopped-flow
stopped-flow
stopped-flow
stopped-flow
stopped-flow
15
17
17
17
18
19
c
c
c
c
c
pH 3.6, 0.1 M H2PO4-, 10 f 130 MPa, 25 °Cb
pH 3.6, 5 mM M H2PO4-, 10 f 130 MPa, 25 °Cb
pH 4.2, 0.1 M H2PO4-, 0.1 f 140 MPa, 25 °Ca
pH 3.6, 5 mM H2PO4-, 0.1 f 140 MPa, 25 °Ca
pH 3.3, 10 mM H2PO4-, 5 f 180 MPa, 3 °Ca
c
a Experiments carried out in Zu¨rich. b Experiments carried out in Erlangen. Slow decomposition of the alkaline peroxynitrite solution occurred
during the experiments; see Discussion. c This work.
the concentrations desired; these solutions were protected from
light and atmospheric carbon dioxide. The acidification to effect
the peroxynitrous acid decomposition was carried out with
phosphoric acid/phosphate buffers of various composition
prepared from analytical grade reagents (Fluka). Ultrapure water
obtained from a Milli-Q unit was used to prepare all aqueous
solutions.
For low ionic strength experiments, the ca. 10 mM sodium
hydroxide solution containing peroxynitrite was mixed with ca.
20 mM phosphoric acid to obtain a pH between 3 and 5. The
actual pH value was determined by mixing equal volumes of 5
mL of the buffer and the peroxynitrite solutions in a small beaker
and measuring the pH with a glass electrode. For high-ionic-
strength experiments, either 0.2 M sodium dihydrogen phos-
phate, or a mixture of 0.2 M sodium dihydrogen phosphate with
ca. 20 mM phosphoric acid, was mixed with peroxynitrite. The
former system resulted in a final pH of 5-6, the latter in a
range of 3-5, as in the low-ionic-strength experiments.
Both high-pressure stopped-flow systems employed are of
well-established designs: one has a sealed internal electrical
drive (Erlangen, Germany),13 whereas the other (Zu¨rich, Swit-
Figure 1. Pressure dependence of the rate constant for the decay of
peroxynitrous acid at low ionic strength (5 mM). Squares: 25 °C,
zerland) has an external pneumatic drive (Hi-Tech HPSF-56,
Salisbury, U.K.) according to a design by the group of Prof. A.
Merbach at EPFL (Lausanne, Switzerland).14 Both instruments
are fully thermostated. Both systems allow a delay of 30 min
prior to measurement, after the pressure increase, to dissipate
the heat generated during adiabatic compression. The peroxy-
nitrite concentration after mixing was kept in the range -100-
600 µM to ensure a reasonable absorbance signal for peroxy-
nitrous acid at the observation wavelength of 260 nm. During
one series of experiments (in Erlangen), substantial decay of
the alkaline peroxynitrite solutions was observed inside the
stopped-flow apparatus. This decay may have been caused by
contact with the drive-syringe piston seals, which are composed
of a Teflon and bronze material. The initial concentration
dropped from 600 to 300 µM during a set of experiments. In
the HI-Tech apparatus, the seals of which are made of Teflon
only, the peroxynitrite was stable for hours, and the initial
peroxynitrite concentration could be kept at 100-150 µM.
Pressure ranges for the various experiments carried out at 25
°C described here were 50-150 MPa (Zu¨rich, May 2001, C.
Thomas), 10-130 MPa (Erlangen, July 2001, R. Kissner and
M. S. A. Hamsa), and 0.1-140 MPa (Zu¨rich, December 2001,
R. Kissner). The pressure range in an experiment at 3 °C
(Zu¨rich, December 2002, R. Kissner) was 5-180 MPa.
peroxynitrite 120 µM, final pH ) 3.6, ∆V‡ ) +6.6 ( 1.0 cm3 mol-1
.
Circles: 25 °C, peroxynitrite 300-600 µM, final pH ) 3.6, ∆V‡ )
+7.8 ( 1.1 cm3 mol-1. k0 ) 1.15 s-1 at 25 °C. Error bars represent the
95% confidence interval for an additional determination.
were found (Table 1, Figure 1). As there is agreement that, at
lower temperature, there is a quantitative conversion of peroxy-
nitrous acid to nitrate, we also carried out experiments at 3 °C
and found an activation volume of 4.8 cm3 mol-1 (Figure 2).
These results are summarized along with available literature data
in Table 1. It should be noted that the literature data15-19 were
selected so as to reflect the currently preferred data as quoted
by the individual authors. For example, an earlier value16 of 2
cm3 mol-1 is not included because the measurements were
carried out at a pH too close to the pKa of peroxynitrous acid.
For the determination of the activation volumes, two different
instrumental techniques were employed. In one method, the
peroxynitrous acid is generated through pulse radiolysis of a
nitrate solution, or of a solution that contains a mixture of
formate and nitrite. In the other method, a stable alkaline
peroxynitrite solution is acidified in situ in a stopped-flow
instrument.
From the data in Table 1, it is clear that the data from the
pulse-radiolysis studies yield an average activation volume of
10.2 ( 1.1 cm3 mol-1. Stopped-flow data from in situ
acidificaton of peroxynitrite range from 6 to 8, with an average
value of 6.7 ( 0.9 cm3 mol-1. Hurst and co-workers recently
At 260 nm and 25 °C we observed the decay of peroxynitrous
acid near pH 4 at pressures up to 150 Mpa. Both at low and
high ionic strength, activation volumes of 6 to 8 cm3 mol-1

Letters
J. Phys. Chem. A, Vol. 107, No. 51, 2003 11263
on the basis of the reported activation volumes, and a more
detailed mechanistic interpretation based on these values is, at
present, pure speculation. Careful ab initio studies may provide
predictions of volume changes associated with the two proposed
reaction mechanisms and allow more detailed interpretation of
the data.
Acknowledgment. We gratefully acknowledge financial
support from the Deutsche Forschungsgemeinschaft and the
ETH Zu¨rich.
References and Notes
(1) Systematic names: O2•-, dioxide(•1-); NO•, oxidonitrogen(•);
NO2-, dioxonitrate(1-); NO3-, trioxonitrate(1-); ONOO-, oxoperoxoni-
trate(1-). The trivial names superoxide, nitrogen monoxide, nitrite, nitrate,
and peroxynitrite, respectively, are allowed. Leigh, G. J., Ed. Nomenclature
of Inorganic Chemistry; Blackwell Scientific Publications: Oxford, U.K.,
1990. Koppenol, W. H. Pure Appl. Chem. 2000, 72, 437-446.
(2) Nauser, T.; Koppenol, W. H. J. Phys. Chem. A 2002, 106, 4084-
4086.
(3) Lymar, S. V.; Hurst, J. K. J. Am. Chem. Soc. 1995, 117, 8867-
Figure 2. Pressure dependence of the rate constant for the decay of
peroxynitrous acid at different temperatures. Squares: 25 °C, ionic
strength 0.1 M, peroxynitrite 150 µM, final pH ) 4.2, ∆V‡ ) +6.0 (
0.5 cm3 mol-1. Circles: 3 °C, ionic strength 0.1 M, peroxynitrite 135
µM, final pH ) 3.8, ∆V‡ ) +4.8 ( 0.2 cm3 mol-1. k0 ) 0.10 s-1 at
3 °C. Error bars represent the 95% confidence interval for an additional
determination.
8868.
(4) Meli, R.; Nauser, T.; Latal, P.; Koppenol, W. H. J. Biol. Inorg.
Chem. 2002, 7, 31-36.
(5) Mere´nyi, G.; Lind, J.; Goldstein, S.; Czapski, G. J. Phys. Chem. A
1999, 103, 5685-5691.
(6) Coddington, J. W.; Hurst, J. K.; Lymar, S. V. J. Am. Chem. Soc.
1999, 121, 2438-2443.
(7) Gerasimov, O. V.; Lymar, S. V. Inorg. Chem. 1999, 38, 4317-
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reported stopped-flow data giving rise to an average value of
(8) Richeson, C. E.; Mulder, P.; Bowry, V. W.; Ingold, K. U. J. Am.
Chem. Soc. 1998, 120, 7211-7219.
9.7 ( 1.4 cm3 mol-1 19
.
There is one activation volume in Table 1 that requires
comment. At 3 °C a value of 4.8 ( 0.2 cm3 mol-1 is reported.
At low temperature, there is nearly quantitative conversion of
peroxynitrous acid to nitric acid without formation of nitrite
and dioxygen.10 In this case, homolysis does not seem to be
the likely reaction pathway.
(9) Koppenol, W. H.; Moreno, J. J.; Pryor, W. A.; Ischiropoulos, H.;
Beckman, J. S. Chem. Res. Toxicol. 1992, 5, 834-842.
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All in all, the average values quoted above are, indeed, not
very disparate when one considers that different laboratories,
using different experimental techniques, different peroxynitrite
preparations, and different reaction media were employed. Thus,
it is safe to conclude that the conversion of peroxynitrous to
nitric acid is characterized by a moderately positive activation
volume. What does this mean in terms of the mechanism of
the conversion reaction? It is reasonable to expect that the
mechanism involving rotation around the N-O bond followed
by intramolecular HO transfer requires a small but moderate
volume increase in the transition state, i.e., in line with the lower
numbers reported in Table 1.20 On the other hand, the suggested
homolysis mechanism is expected to require a substantial
volume increase in going to the transition state, i.e., in line with
the higher numbers reported in Table 1.17-20 This means that
the range of experimental values in Table 1 does not enable
definitive discrimination between the two possible mechanisms
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40, 528-532.
(19) Lymar, S. V.; Khairutdinov, R. F.; Hurst, J. K. Inorg. Chem. 2003,
in press.
(20) (a) Drljaca, A.; Hubbard, C. D.; van Eldik, R.; Asano, T.;
Basilevsky, M. V.; le Noble, W. J. Chem. ReV. 1998, 98, 2167-2289. (b)
High-Pressure Chemistry: Synthetic, Mechanistic and Supercritical Ap-
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