Reaction of ruthenium nitrosyl complexes with superoxide

Article abstract of DOI:10.1016/j.poly.2012.11.021

The reaction between the ruthenium nitrosyl complexes trans-[Ru ^II(NO)L₁L₂]ⁿ⁺, where L₁ = (NH₃)₄ and L₂ = imN, nic, 4-pic and P(OEt)₃ or L₁/L₂ = Hedta, with superoxide (O₂^-) have been probed in aqueous medium at (25 ± 0.1) °C by UV-Vis, DPV and EPR spectroscopies. The reaction involves one electron transfer from O₂^- to trans-[Ru ^II(NO)L₁L₂]ⁿ⁺ yielding trans-[Ru^II(H₂O)L₁L₂] ⁿ⁺ and nitric oxide (NO) as main reaction products. Using cytochrome c as a probe for superoxide and a competitive kinetic approach, the apparent bimolecular rate constant for the reaction between trans-[Ru ^II(NO)L₁L₂]ⁿ⁺ and O ₂^- have been determined to be in the range of (6.3 ± 0.5) × 10³ (L₁ = (NH₃) ₄; L₂ = 4-pic) to (5.8 ± 0.2) × 10 ⁶ (L₁ = (NH₃)₄; L₂ = P(OEt)₃) M^-1 s^-1. For L₁ = (NH ₃)₄ and L₂ = P(OEt)₃ the peroxynitrite formation as a by-product of O₂^- and NO reaction was detected using tyrosine as a probe monitoring the formation of 3-nitro-tyrosine by HPLC-DAD. These nitrosyl compounds can be activated by superoxide, thus holding radical scavenger potential in vivo, besides being useful to modulate the local NO levels.

Full text of DOI:10.1016/j.poly.2012.11.021

Polyhedron 50 (2013) 328–332
Contents lists available at SciVerse ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Reaction of ruthenium nitrosyl complexes with superoxide
⇑
Gustavo Metzker, Daniel Rodrigues Cardoso, Douglas Wagner Franco
Institute of Chemistry of São Carlos, University of São Paulo, Av. Trabalhador São-Carlense 400, Centro. São Carlos-SP, Brazil
a r t i c l e i n f o
a b s t r a c t
The reaction between the ruthenium nitrosyl complexes trans-[RuII(NO)L1L2]n+, where L1 = (NH3)4 and
L2 = imN, nic, 4-pic and P(OEt)3 or L1/L2 = Hedta, with superoxide (O2Åꢀ) have been probed in aqueous
medium at (25 0.1) °C by UV–Vis, DPV and EPR spectroscopies. The reaction involves one electron trans-
fer from O2Åꢀ to trans-[RuII(NO)L1L2]n+ yielding trans-[RuII(H2O)L1L2]n+ and nitric oxide (NO) as main reac-
tion products. Using cytochrome c as a probe for superoxide and a competitive kinetic approach, the
Article history:
Received 4 July 2012
Accepted 19 November 2012
Available online 29 November 2012
apparent bimolecular rate constant for the reaction between trans-[RuII(NO)L1L2]n+ and O2 have been
Åꢀ
Keywords:
Ruthenium
Nitric oxide
Superoxide
determined to be in the range of (6.3 0.5) ꢁ 103 (L1 = (NH3)4; L2 = 4-pic) to (5.8 0.2) ꢁ 106
(L1 = (NH3)4; L2 = P(OEt)3) Mꢀ1 sꢀ1. For L1 = (NH3)4 and L2 = P(OEt)3 the peroxynitrite formation as a by-
Åꢀ
product of O2 and NO reaction was detected using tyrosine as a probe monitoring the formation of
3-nitro-tyrosine by HPLC-DAD. These nitrosyl compounds can be activated by superoxide, thus holding
radical scavenger potential in vivo, besides being useful to modulate the local NO levels.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
(Trypanosoma cruzi and Leishmania major) and cancer [27]. Further-
more, in a reductive environment, as found under hypoxia condi-
tions, where the iNOS is inhibited [28], these compounds could
act as an alternative and controlled NO source.
With an aim to better understand the chemical reactivity of
these ruthenium nitrosyl complexes when administrated in biolog-
ical medium, this paper describes the reactivity of the trans-
[RuII(NO)L1L2]n+ towards superoxide in aqueous medium.
Superoxide (O2Åꢀ) is widely produced by mammalian cells,
mostly by the mitochondrial respiration process [1–3]. It is esti-
mated that 1–3% of all oxygen uptake by mammalian respiration
is converted into superoxide [1]. Although, under conditions of oxi-
dative stress, the consumption of molecular oxygen and conse-
Åꢀ
quently the production of O2 increases dramatically [1,4]. As a
consequence of its continuous production and reactivity, superox-
ide is a key molecule in human physiology [1,5].
2. Materials and methods
Nitric oxide (NO) is a widely studied molecule due to its role in
human physiological pathways [6–12]. Nitric oxide reacts with
superoxide at rate constant close to the diffusion limit in water,
generating peroxynitrite (ONOOꢀ) [13–15]. This is a high reactive
molecule and may be responsible for cells damages and oxidative
stress [16–21]. From this perspective the control of the local NO
concentration becomes very important.
2.1. Reagents and solutions
All reagents were purchased from Sigma-Aldrich, Fluka or
Merck and used as received. A 5.0 ꢁ 10ꢀ2 M Phosphate buffer
pH = (7.2 0.1)
(18.2 M
l = 0.1 M was prepared using high purity water
cm) from a Milli-Q purification system (Millipore, Bed-
X
Metal based nitric oxide carriers, chemically or photochemically
activated, have been deserving attention as much as NO itself,
since the controlled delivering or uptake of NO is required [22–
26]. Ruthenium nitrosyl complexes, trans-[RuII(NO)L1L2]n+, where
L1 = (NH3)4 and L2 = N-heterocyclic or phosphane ligands [23];
L1/L2 = Hedta are able to dissociate NO or HNO after reduction of
coordinated NO+ by reductants such as ascorbic acid and thiols
(e.g. GSH) [23]. As consequence of the NO releasing ability, these
compounds were tested in vitro and in vivo as candidate drugs
for vasodilatator and as pro-drugs against tripanosomatides
ford, MA). Horse-heart ferricytochrome c was purified using a
Sephadex G-25 column (0.5 ꢁ 15 cm) [29]. The concentration of
the purified ferricytochrome c (Cyt c) solution was calculated by
UV–Vis spectroscopy [30].
2.2. Instrumentation
UV–Vis spectra were recorded on a Hitachi U-3501 spectropho-
tometer using 1.00 cm quartz cells. Samples were irradiated with a
Nd:YAG LASER (Continuum, model Surelite-II) operating in the
third harmonic (355 nm) pumping an optical parametric oscillator
(OPO) system (Continuum, model 10-05-00 SSP-4) adjusted to
440 nm using a wavelength meter (Coherent, model Wavemate).
⇑
Corresponding author. Tel.: +55 16 3373 9970; fax: +55 16 3373 9976.
E-mail address: douglas@iqsc.usp.br (D.W. Franco).
0277-5387/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2012.11.021

G. Metzker et al. / Polyhedron 50 (2013) 328–332
329
A power meter (Coherent, Lasermate-P) measured the average en-
ergy per pulse. The selective detection of nitric oxide was carried
out chronoamperometrically using a NO selective electrode (Inno-
vative Instruments Inc., model inNO-T). The electrode was polar-
ized in water for 12 h before the measurements. Electron
paramagnetic resonance (EPR) experiments were carried out on a
Bruker ESP300E spectrometer at X-Band frequency. Measurements
were carried out at 100 K using a variable temperature system
(Euroterm, model B-VT 2000). For spectra calibration, a sealed cap-
illary containing a small amount of solid DPPHÅ (g = 2.0036) was
introduced into the samples tubes and the spectra were recorded.
HPLC analyses were carried out in a LC-10AD-VP system coupled to
a SPD-M10AVD UV–Vis diode array detector (Shimadzu). Differen-
tial pulse voltammetry (DPV) measurements were performed on
an EG&G PAR model 264A, using a glassy carbon electrode as a
working electrode, a saturated calomel electrode (SCE) as reference
and a platinum plate as auxiliary electrode. Solutions of CF3COOH/
was used as a competitor (kCyt c = 1.4 ꢁ 106 Mꢀ1 sꢀ1 for superoxide)
[43] and the reaction course was monitored by UV–Vis spectros-
copy. Concentrations of ruthenium nitrosyl complexes ranged over
the range of 1.0 ꢁ 10ꢀ2 to 1.0 ꢁ 10ꢀ5 M depending on the nature of
the ligand L. Control experiments without addition of the ruthe-
nium complexes were loaded between each sample. By probing
the absorbance at 550 nm for ferricytochrome c in the control
experiments and in the samples containing the ruthenium nitrosyl
complexes, the bimolecular apparent rate constant (kapp) for the
reaction were calculated by plotting (S/1 ꢀ S) kCyt c [Cyt c] versus
[trans-[RuII(NO)L1L2]n+] where S is the percentage of inhibition of
the ferricytochrome c reduction by the ruthenium nitrosyl com-
plexes as follows:
ꢀ
ꢁ
h
i
S
nþ
kCyt cjCyt cj ¼ kapp trans-½RuIIðNOÞL1L2ꢂ
1 ꢀ S
CF3COONa, pH = (5.5 0.1)
l = 0.1 M were used as the background
2.6. Peroxynitrite detection
electrolyte. Experimental conditions for DPV were scan rate of
20 mV/s and pulse height of 100 mV. Both trans-[RuII(NO)(NH3)4
(P(OEt)3)]3+ and complex trans-[RuII(H2O)(NH3)4(P(OEt)3)]2+ are
robust species in aqueous acidic solutions and therefore the
trans-[RuII(NO)(NH3)4(P(OEt)3)](PF6)3 salt was used as internal stan-
dard when quantification was necessary. The measured potentials
were converted and reported as normal hydrogen electrode (NHE).
31P NMR spectra were obtained in a Bruker AVANCE (400 MHz) spec-
trometer, using a 5 mm probe. The counter-ion hexafluorophosphate
was used as internal reference (PF6ꢀ; d = ꢀ144 ppm).
Peroxynitrite detection during the reaction of superoxide with
the ruthenium nitrosyl complexes was carried out using tyrosine
as a probe and analyzing its nitration product, 3-nitro-tyrosine
[44–46] by HPLC-DAD detection. Solutions of ruthenium nitrosyl
complexes were photolyzed in the presence of L-tyrosine
(2.7 ꢁ 10ꢀ3 M) in the same photolysis conditions described before.
Chromatography analyzes were performed using a 5.0 ꢁ 10ꢀ2
M
phosphate buffer (KH2PO4; pH = 3.0 0.1) and methanol (95:5 v/v).
A Waters Spherisorb S5 ODS (250 ꢁ 2.0 mm i.d. and 5
lm particle
size), chromatographic column was used at the flow rate of 0.3 mL/
min and at a temperature of 22 °C. Under these experimental con-
ditions, the detection limit for 3-nitro-tyrosine was determined as
2.0 ꢁ 10ꢀ7 M, being comparable with the one reported in the
2.3. Synthesis of ruthenium amines nitrosyl complexes
Ruthenium
nitrosyl
complexes
trans-[RuII(NO)L1L2](X)n
(L1 = (NH3)4 and L2 = 4-picoline (4-pic), nicotinamide (nic), imidaz-
ole (imN), and triethylphosphite (P(OEt)3); and L1/L2 = Hedta,
X = hexafluorophosphate (PF6ꢀ) or tetrafluoroborate (BF4ꢀ), and
their synthetic precursors were prepared and characterized as
described in the literature [31–37]. When necessary, the
solutions were degassed using purified and dried argon and
manipulations were carried out using standard inert atmosphere
techniques [38].
literature [46]. Photolyzed solution (20 lL) was injected and
the 3-nitro-tyrosine formation was followed at 365 nm. 3-
Nitro-tyrosine is not the only product of the reaction between tyro-
sine and peroxynitrite and therefore the result was used only for
semi quantitative purposes. Nevertheless an estimative of the 3-
nitro-tyrosine amount formed was performed by the standard
addition method.
2.4. Generation and detection of superoxide
3. Results and discussion
Superoxide was generated photochemically or enzymatically
(xanthine/xanthine oxidase) as previously described in the litera-
ture [29,39]. For the photochemical generation, air saturated solu-
The reaction of superoxide with the ruthenium nitrosyl com-
plexes was studied by chronoamperometric measurements, DPV
and UV–Vis, 31P NMR, and EPR spectroscopies. Samples were EPR
silent, suggesting that the metal center was not oxidized to Ru(III)
in the course of the reaction.
tions containing 1.0 ꢁ 10ꢀ3
M flavin mononucleotide (FMN),
1.0 ꢁ 10ꢀ2 M of diethylenetriaminepentaacetic acid (DTPA) were
irradiated for 4 min using a Nd:YAG LASER with a repetition-rate
of 1 pulse per second (8 ns duration) with average energy of
2.5 0.9 mJ per pulse. To probe the superoxide generation, the
reduction of a ferricytochrome c solution was monitored using
UV–Vis spectroscopy (Fig. S1) [30]. For the enzymatic assay [29],
1.0 ꢁ 10ꢀ3 M xanthine solutions in phosphate buffer were used.
Xanthine oxidase solutions (0.016 U/mL) were freshly prepared
in the same buffer. After mixing the solutions, the reaction pro-
ceeded for a period of 20 min. Superoxide generation was con-
firmed through the detection of the ferricytochrome c reduction
by UV–Vis [29,30].
After the photochemical generation of superoxide in presence of
trans-[RuII(NO)(NH3)4(P(OEt)3)]3+ the complex trans-[RuII(H2O)
(NH3)4(P(OEt)3)]2+ was observed in solution by DPV (Fig. 1, peak
0
a, E0 = 0.70 V versus NHE) [47] and 31P NMR (d = 148 ppm, data
not shown) measurements [46]. After 4 min of photolysis, approx-
imately 30% of the initial amount of trans-[RuII(NO)(NH3)4
0
(P(OEt)3)]3+ (Fig. 1, peak b, E0 = 0.12 V versus NHE) was converted
into the aqua complex. Nitric oxide evolution was not detected
chronoamperometrically (Fig. S2) using a selective NO electrode.
Using DTPA/FMN/light as superoxide source and cytochrome c
reduction as a probe [43], the concentration of superoxide in solu-
tion was calculated as being approximately 9.6 ꢁ 10ꢀ6 M (Fig. S1).
As shown by the DPV response, the concentration of trans-[RuII-
(H2O)(NH3)4(P(OEt)3)]3+ formed was equal to 9 ꢁ 10ꢀ6 M. Thus,
the reaction of superoxide with the trans-[RuII(NO)(NH3)4
(P(OEt)3)]3+ yielded 92% of the predicted amount of the aqua
complex for a stoichiometric reaction. The phosphite complex
was chosen to be studied in more details because its aqua species
2.5. Competitive kinetic
A competitive kinetic experimental method [40–42] was used
to estimate the bimolecular apparent rate constants for the reac-
tion between superoxide and the ruthenium nitrosyl complexes.
A solution at concentration of 2.0 ꢁ 10ꢀ5 M of ferricytochrome c

330
G. Metzker et al. / Polyhedron 50 (2013) 328–332
ruthenium nitrosyl complexes are given in Table 1. The plots for
kapp calculation for the other ruthenium nitrosyls herein studied
are in the Supplementary Material section (Fig. S3).
The data of Table 1 show that the kapp values were influenced by
the nature of the ligands L1 and L2. A clear trend was not observed
between the nitrosonium reduction potential of the [RuNO]3+/2+
couple or the
their respective values of kapp
m
(NO+) (Table S1) in all of the title compounds and
.
Since superoxide and nitric oxide are present in solution, the
possibility of peroxynitrite formation was also investigated, using
tyrosine nitration as probe [46]. Under the experimental conditions
used, the formation of ONOOꢀ was observed only for the complex
trans-[RuII(NO)(NH3)4(P(OEt)3)]3+. The utilization of tyrosine for
peroxynitrite identification is extensive discussed in the literature
[44–46,50]. Since the reaction medium is not anaerobic, the dis-
solved CO2 in the solution can participate of the reaction. As dis-
cussed by Lancaster [45], only one third of the peroxynitrite
produced can nitrosilate the tyrosine, through the generation of
Å
NO2 and CO3Åꢀ. Also, as proposed by Houk and co-workers, there
is more than one possible pathway for tyrosine nitration [50]. A
simplified scheme of these reactions is presented in the Fig. S4.
Thus, in the present experimental approach, tyrosine was used
only for identification and to estimate the amount of peroxynitrite
formed during the reaction. An estimative of 3-nitro-tyrosine
amount formed, carried out by HPLC-DAD, suggest that when the
Fig. 1. Differential pulse voltammogram of the photolyzed solution containing
trans-[RuII(NO)(NH3)4(P(OEt)3)]3+ DTPA and FMN. Dotted line: before irradiation;
solid line: after 4 min of photolysis, peak a and b: aqua and nitrosyl complexes
l = 0.1 M CF3COOH/CF3COONa;
scan rate: 20 mV/s; pulse height: 100 mV; T = (25 0.1) °C.
respectively. CRu = 3.0 ꢁ 10ꢀ5 M; pH = (5.5 0.1),
concentration
of
trans-[RuII(NO)(NH3)4(P(OEt)3)]3+
was
is more stable in acidic solution regarding to trans-[RuII(H2O)L1L2]n+
where L2 = N-heterocycles [23].
1.25 ꢁ 10ꢀ4 M, the concentration of 3-nitro-tyrosine in solution
was 1.7 ꢁ 10ꢀ5 M.
The kinetic measurements were carried out by monitoring
absorbance changes at 550 nm and the kinetics calculations were
performed as described in Section 2.5. The ruthenium nitrosyl
complexes spectra were discussed previously [48] and they photo-
chemical behavior as well [49]. According to these data these com-
pounds are not photoactive in the wavelength utilized (kirrad = 440
nm) precluding eventual side reactions. Fig. 2 shows the spectra of
the reduction of cytochrome c by superoxide and its inhibition
when the solution was photolyzed in the presence of ruthenium
nitrosyl complexes.
The superoxide may act as an oxidant or reductant, depending
on the reaction conditions. The reported pKa value for the perhydr-
Å
oxyl radical (HO2 ) is 4.7 [51]. Thus, in most of the cases, especially
in biochemical processes, the predominant form of superoxide is
the deprotonated form, O2Åꢀ. Since the redox potential for superox-
ide is reported to be ꢀ0.16 V [52], the reduction of the coordinated
NO+ is thermodynamically feasible (Table S1).
Molecular orbitals analysis of the ruthenium nitrosyl complexes
indicates that the LUMO orbitals have a predominant NO+ ligand
character (for example, L = py ꢀ LUMO: 28% Ru, 67% NO+, 3% py;
LUMO + 1: 28% Ru, 70% NO+) [48]. Thus, it is reasonable to suppose
that the reduction of these complexes by superoxide occurs prefer-
entially on the nitrosonium ligand. On this perspective, and taking
in account the experimental data presented herein, the reaction
between superoxide and the ruthenium nitrosyl complexes would
be coherent with the sequence of reactions:
The bimolecular apparent rate constant (kapp) was calculated
using the Fig. 2 data, the control experiment (Fig. S1) and the equa-
tion described in the Section 2.5. The values of kapp obtained for the
kapp
trans-½RuIIðNOÞL1L2ꢂnþ þ O2 ! trans-½RuIIðNOÞL1L2ꢂðnꢀ1Þþ þ O2
Åꢀ
ð1Þ
trans-½RuIIðNOÞL1L2ꢂðnꢀ1Þþ þ H2O k!—NO trans-½RuIIðH2OÞL1L2ꢂðnꢀ1Þþ þ NO
ð2Þ
The ruthenium nitrosyl complexes can undergo to a second
chemical or electrochemical reduction at the coordinated nitric
oxide, yielding HNO [53]. However, the second reduction reaction
Table 1
Apparent bimolecular rate constants (kapp) for superoxide reaction with ruthenium
nitrosyl complexes in aqueous medium at (25 0.1) °C.a
L1/L2
kapp (Mꢀ1 sꢀ1
)
b
(NH3)4/P(OEt)3
(NH3)4/imN
(NH3)4/nic
(NH3)4/4-pic
Hedta
(5.8 0.2) ꢁ 106
(1.3 0.1) ꢁ 105
(7.2 0.5) ꢁ 104
(6.3 0.5) ꢁ 103
(3.2 0.3) ꢁ 104
Fig. 2. UV–Vis spectra of the reaction between trans-[RuII(NO)(NH3)4((POEt)3)]
(PF6)3 with superoxide using cytochrome c as probe. a: CRu = 3.5 ꢁ 10ꢀ5 M; b:
CRu = 5.0 ꢁ 10ꢀ5 M; c: CRu = 6.5 ꢁ 10ꢀ5 M; d: CRu = 7.5 ꢁ 10ꢀ5 M; e: CRu = 8.5
a
pH = (7.2 0.1).
pH = (5.5 0.1).
b
ꢁ 10ꢀ5 M; pH = (5.5 0.1)
l
= 0.1 M; T = (25 0.1) °C. CCyt c = 2.0 ꢁ 10ꢀ5 M.

G. Metzker et al. / Polyhedron 50 (2013) 328–332
331
is not possible on thermodynamic grounds with superoxide, since
nitrosyl complexes and superoxide is fast, the formation of ONOOꢀ
(Eq. (3)) would be dependent of the NO concentration generated
from in solution.
00
the E
value for the ruthenium tetraamine complexes is
ðNO0=HNOÞ
more negative than ꢀ0.50 V [53]. Furthermore, the back reaction
of the Eq. (2), is not favorable since Keq ꢃ 30 Mꢀ1 [23].
Among the compounds studied herein, the complex trans-[RuII
(NO)(NH3)4(P(OEt)3)]3+ exhibited the highest values of kapp and
k–NO, which would generate the major NO concentration in solu-
tion, and thus turning more feasible the formation of ONOOꢀ in
an appreciable extension. The high limit for the NO concentration
in our experiments would be equal to the one of the produced
superoxide (ꢃ1.0 ꢁ 10ꢀ5 M). Despite the high value for reaction
between superoxide and NO (k ꢃ 109 Mꢀ1 sꢀ1) [14,15] we must
to have in mind that the reaction between NO and O2Åꢀ is first order
in both reactants and the half life for superoxide ion in solution
small due to its dismutation [62].
The apparent bimolecular rate constant (kapp), Table 1, are in the
range of (6.3 0.5) ꢁ 103 to (5.8 0.2) ꢁ 106 Mꢀ1 sꢀ1. These values
are in agreement with those reported for other d6 low spin metal
complexes and metalloproteins, such as cytochrome
c [54].
Regarding the reaction of superoxide with other nitric oxide do-
nors, Aleryani and co-workers [55] reported that the values for
the specific rate constants for S-nitrosocysteine and S-nitrosogluta-
thione were (7.69 0.64) ꢁ 104 and (1.28 0.05) ꢁ 104 Mꢀ1 sꢀ1
,
respectively. These values are in a good agreement with the ones
measured for the ruthenium nitrosyl complexes described herein.
Superoxide can also act as a nucleophilic agent. Ruthenium
nitrosyl complexes are susceptible to nucleophilic attack on the
coordinated NO+ by OHꢀ or thiols (RSꢀ) to yield the species,
trans-[RuII(NO2)(NH3)4L](nꢀ1)+ and trans-[RuII(N(O)SR)(NH3)4L]n+
respectively, as described in the literature [56,57]. As mentioned
before, the NO liberation was observed by chronoamperometric
measurements using a selective NO electrode suggesting that the
nucleophilic attack of superoxide on trans-[RuII(NO)L1L2]n+ through
the formation of trans-[RuII(NOOO)L1L2](nꢀ1)+ is unlikely to occur,
at least to any appreciable extent. If the formation of an intermedi-
ate species with superoxide and coordinated NO+ has occurred, the
products would probably be peroxynitrite and a Ru(III). In our
experiments, no paramagnetic species was detected by EPR during
the experiments, hence suggesting that the nucleophilic attack at
the coordinate NO+ did not occur.
For the other complexes the values of kapp and k–NO are much
lower than those for the L2 = P(OEt)3 and therefore the available
concentration of free NO in solution is lower. These observations
are in agreement with the experimental data, since no 3-nitro-
tyrosine formation was detected when L1 = (NH3)4; L2 = imNand
L1/L2 = Hedta (k–NO = 0.16 and 7.3 ꢁ 10ꢀ3 sꢀ1
; kapp = (1.3 0.1)
ꢁ 105 and (3.2 0.3) ꢁ 104 Mꢀ1 sꢀ1, respectively). However, under
situations where superoxide production is continuous and the con-
centration of this molecule is relatively high, as found for example
inside mitochondria [2,3] the formation of peroxynitrite would be
possible. It is interesting to recall that the concentration of super-
oxide in tissues may range from ca. 1.0 ꢁ 10ꢀ9 to ca 1.0 ꢁ 10ꢀ6 M in
basal or in inflammatory conditions, respectively [1,10].
Two other possible biological reductants for the ruthenium
nitrosyls are cysteine (Cys) (pKaCys = 8.3, E00
= ꢀ0.245 V,
Cysteine=Cystine
According to Weinstock [58], the reaction of transition metal
complexes with superoxide proceeds by an outer-sphere electron
transfer mechanism. It is interesting to recall at this point that
the kapp values obtained for the reaction of superoxide with the
ruthenium nitrosyl complexes, and the values calculated and re-
ported in the literature for the complexes [RuIII(edta)(H2O)]ꢀ and
trans-[RuIII(NH3)6]3+ [58] are in the same order of magnitude.
Ruthenium nitrosyl complexes are robust concerning substitution
reactions (the same is not true for photochemical reactions) in
aqueous medium for at least one week [23]. As substitution reac-
tions do not take place for these complexes on the time scale of
the reactions here described, it is feasible to suppose that the
reduction of the NO+ by superoxide follows an outer-sphere elec-
tron transfer mechanism.
pH = 7.0) [63] and NADPH (E0NADP=NADPH = ꢀ0.324 V, pH = 7.0) [64],
which are found in the millimolar concentration range [65]. For
cysteine, more than 95% is in the protonated form under physiolog-
ical conditions, with the concentration of the active reductant,
Cysꢀ, ca. 1.0 ꢁ 10ꢀ5 M. The specific rate constant for the reaction
between Cysꢀ and NADPH with ruthenium nitrosyl complexes
are 1.6 ꢁ 105 and 2.0 ꢁ 101 Mꢀ1 sꢀ1, respectively [56,66]. Compar-
ing the rate constants and the concentrations of the species
NADPH, Cysꢀ and superoxide, under inflammatory conditions or
circumstances where the concentration of superoxide is in the
micromolar scale, it is reasonable to infer that superoxide along
with Cysꢀ would be candidates to act as important reductants for
ruthenium nitrosyl complexes in biological systems.
Åꢀ
After the reduction of nitrosonium ligand by O2 and conse-
Another important reaction involving free nitric oxide and
superoxide is the peroxynitrite formation. Under our experimental
conditions, peroxynitrite could be formed after the reactions of
Eqs. (1) and (2), by the interaction of nitric oxide with superoxide
(Eq. (3)).
quently NO liberation the aqua species trans-[RuII(H2O)L1L2](nꢀ1)+
is formed. NO itself is a multi-target biologically active species,
but the aqua species of transition metal complexes is also active
as discussed in the literature [67–69]. The aquation step of the
transition metal complexes, as the platinum and the ruthenium
species, is a key step for their biological properties [67–69], since
the aquo species can bind on DNA [68–70] and other proteins like
albumin [69]. Also trans-[Ru(H2O)(NH3)5]n+, where Ru is in the
oxidation state II or III, trans-[RuII(H2O)(NH3)5(pz)]2+, and [RuII-
(H2O)(edta)] showed anti-cancer activity [69]. Thus, the complexes
trans-[RuII(NO)L1L2]n+ would exhibit two possible different
pathways of action in vivo, as consequence of the reaction with
NO þ O2 ! ONOOꢀ
ð3Þ
Åꢀ
The formation of peroxynitrite was only observed when trans-
[RuII(NO)(NH3)4(P(OEt)3)]3+ reacts with superoxide. The detection
of 3-nitro-tyrosine by HPLC-DAD confirmed the formation of per-
oxynitrite during the reaction. For all other ruthenium nitrosyl
complexes studied, peroxynitrite was not detected. The literature
reports that the presence of superoxide dismutase (SOD) or
[FeIII(edta)]ꢀ increases the yield of the tyrosine nitration [59].
However, in our experiments, even SOD or [FeIII(edta)]ꢀ were not
used since they can react with superoxide (k = 2 ꢁ 109 [60] and
k = 1.9 ꢁ 106 Mꢀ1 sꢀ1 [61], respectively) diminishing the yield of
the ruthenium nitrosyl complexes reduction.
O2 yielding NO and trans-[RuII(H2O)L1L2](nꢀ1)+ both biologically
Åꢀ
active.
4. Conclusions
The failure of ONOOꢀ detection for the nitrosyl complexes
(except for L2 = P(OEt)3) can be tentatively explained by taking in
account their respective specific rate constants for NO liberation
(Eq. (2)). Since the measured superoxide concentration is almost
the same in all the experiments and the reaction between the
Ruthenium nitrosyls complexes react with superoxide by reduc-
tion of the coordinated NO+, yielding NO and the corresponding
aqua complex as products. The apparent bimolecular rate constants
found for this reaction are within the same range reported for other
metal complexes described in the literature. Experimental evidence

332
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trans-[RuII(NO)(NH3)4(P(OEt)3)]3+, which suggest that the specific
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Acknowledgments
The authors acknowledge the Brazilian agencies FAPESP, CAPES
and CNPq for their financial support.
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