Reactions of Manganese Porphyrins with Peroxynitrite and Carbonate Radical Anion*
Gerardo Ferrer-Sueta
,
Darío Vitturi
¶,
Ines Batini
-Haberle || **,
Irwin Fridovich || 
,
Sara Goldstein 
,
Gidon Czapski 
and
Rafael Radi ¶¶ ||||
From the
Laboratorio de Fisicoquímica
Biológica, Facultad de Ciencias, Universidad de la República,
Montevideo 11400, Uruguay, ||Department of
Biochemistry, Duke University Medical Center, Durham, North Carolina 27710,

Department of Physical Chemistry, The
Hebrew University, Jerusalem 91904, Israel, and
¶¶Departamento de Bioquímica,
Facultad de Medicina, Universidad de la República, Av. Gral. Flores
2125, Montevideo 11800, Uruguay
Received for publication, December 31, 2002
, and in revised form, April 15, 2003.
 |
ABSTRACT
|
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We have studied the reaction kinetics of ten manganese porphyrins,
differing in their meso substituents, with peroxynitrite
(ONOO) and carbonate radical anion
(
)
using stopped-flow and pulse radiolysis, respectively. Rate constants for the
reactions of Mn(III) porphyrins with ONOO ranged from 1
x 105 to 3.4 x 107
M1 s1 and correlated
well with previously reported kinetic and thermodynamic data that reflect the
resonance and inductive effects of the substituents on the porphyrin ring.
Rate constants for the reactions of Mn(III) porphyrins with
ranged from 2 x 108 to 1.2 x 109
M1s1 at pH
8.5
and increased with pH as a consequence of the ionization of the complexes.
Mn(II) porphyrins reacted with
with rate constants ranging from 1 x 109 to 5 x
109 M1s1
at pH 10.4. Hence, fast scavenging of ONOO and
by manganese porphyrins could occur in vivo because of the catalytic
reduction at the expense of a number of cellular reductants. Additionally, we
determined the pKa of the axial water molecules
of the Mn(III) complexes at pH 7.513.2 by spectrophotometric titration.
Results were consistent with two acid-base equilibria for most of the
complexes studied. The pKa values also correlated
with the resonance and inductive effects of the substituents. The correlations
of E
with the rate constants with ONOO and
with the pKa values display a deviation from
linearity when N-alkylpyridinium substituents included
N-alkyl moieties longer than ethyl, which is interpreted in terms of
a decrease in the local dielectric constant.
 |
INTRODUCTION
|
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Metalloporphyrins catalyze numerous redox reactions
(1); in particular, manganese
porphyrins have been used as redox catalysts in several model systems relevant
to biochemistry, for instance, as superoxide dismutase
(2,
3) and catalase
(4) mimics. Some of the
N-alkylpyridinium substituted complexes afforded protection of
superoxide dismutase-deficient Escherichia coli from O2
toxicity (5) and in several
rodent models of transient brain ischemia
(6,
7), diabetes
(8), sickle cell disease
(9), and radiation injury
(10). Moreover,
MnIIITCPP1
has been effective in a number of model studies of oxidative stress-mediated
injury (for a review see Ref.
11) despite having low
superoxide dismutase and catalase activities.
Our group
(1214)
and others (15) have studied
the capability of manganese porphyrins for the catalytic reduction of
peroxynitrite (ONOOH/ONOO), a powerful oxidant that can be
formed in vivo by the reaction of
with ·NO (16,
17). A significant fraction of
the oxidative biochemistry of peroxynitrite is derived from the rapid reaction
of ONOO with CO2 (k = 5.8 x
104 M1s1
at 37 °C
(1820)),
which produces the carbonate radical anion
(
)
and nitrogen dioxide (·NO2) with about 33% yield (see
Equation 1, below), with the
remaining yielding carbon dioxide and nitrate, where
k1b/k1a
2
(2124).
 | (Eq. 1) |
Given the ubiquity of CO2, its high concentration (1 to 2
mM in human tissues), and the reactivity of
(25), a useful peroxynitrite
scavenger needs to out-compete the target molecules and CO2 and/or
be able to efficiently scavenge
.
We have proposed that complexes such as MnIIITM-2-PyP can
efficiently inhibit peroxynitrite-mediated oxidations even in the presence of
CO2 (13). Moreover,
the reaction of MnIIITM-2-PyP with ONOO in the
presence of CO2 produced more oxidation of the metal complex than
expected based on simple competition kinetics
(13), suggesting a probable
reaction of the complex with
.
The possible reaction of Mn(III) porphyrins with
has also been proposed recently
(26) in experiments related to
the effect of bicarbonate on the peroxidase activity of Cu,Zn superoxide
dismutase.
In aerated aqueous solution, the stable oxidation state of manganese
porphyrins used in this study is Mn(III). However, given the low oxygen
tension inside the cell, cellular components, like low molecular weight
reductants (27) and probably
some dehydrogenases (28), can
produce Mn(II) porphyrins and maintain them in the reduced state. Mn(II)
porphyrin chemistry has been studied since the seventies
(2931),
but only recently has its biochemistry begun to be explored
(27). Mn(II) porphyrins may
have a number of advantages over Mn(III) porphyrins with regard to their
scavenging and antioxidant activity. For instance, they could rapidly scavenge
oxidizing radicals such as
and yield innocuous products, or they could reduce strong oxidants like
ONOO or H2O2 via a two-electron
transfer reaction without producing any secondary radicals. This latter
reaction is particularly important if Mn(II) can be regenerated at the expense
of readily available biological reductants
(14).
Carbonate radical anion is long known to radiation chemists
(32,
33) but has only recently
drawn attention of biochemists because of its formation from the reaction of
ONOO with CO2
(1621)
and the effect of bicarbonate on the peroxidase activity of Cu,Zn superoxide
dismutase (34). Carbonate
radical anion is the conjugate base of a strong acid
(pKa < 0)
(35) and a strong oxidant
(36) with a characteristic
spectrum in the visible (
600 = 1860
M1cm1)
(32,
33). Nevertheless, its
reactivity is somewhat selective, and its potential targets in biological
systems include sulfur-containing and aromatic amino acids
(37,
38).
Manganese porphyrins display linear free energy relationships between
ligand or complex properties, e.g. pKa
of pyrrolic nitrogens, E
(Mn(III)/Mn(II)), and rate
constants, such as the catalytic rate constant of
dismutation (5,
39). These linear free energy
relationships break down if N-alkylpyridinium substituted porphyrins
contain alkyl groups longer than ethyl, which has been ascribed to steric and
solvation differences in the metal surroundings
(39). The complexes possess
two axially coordinated water molecules, and they can undergo up to two
ionization steps in the alkaline pH range. Data in the literature with respect
to these ionization reactions are diverse, both in the methods used and in the
results. For instance, MnIIITM-4-PyP displays a single
pKa of 10 according to 1H NMR
(40) but two
pKas at 10.9 and 12.3
(41) or at 8.0 and 10.6
(42) by spectrophotometric
titration.
In what follows, we examine the reduction of ONOO by
Mn(III) porphyrins and the reaction of both Mn(II) and Mn(III) porphyrins with
.
We also use spectrophotometric titration to determine the relevant
pKa values.
 |
EXPERIMENTAL PROCEDURES
|
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ChemicalsMn(III) porphyrins used in this work are listed in
Table I along with their
electric charge at pH 7. MnIIITCPP and MnIIITSPP were
purchased from MidCentury Chemicals, Chicago, IL, and the other porphyrins
were synthesized as described previously
(39). Peroxynitrite was
synthesized from hydrogen peroxide and sodium nitrite in acidic solution
(43). All other chemicals were
commercial. Mn(II) porphyrins were prepared by the reduction of Mn(III)
porphyrins with equimolar dithionite in N2O-saturated solutions
containing 0.1 M carbonate at pH 10.4. Dithionite solution was
prepared in helium-saturated solutions containing 0.1 M carbonate
at pH 10.4, and its concentration was assessed immediately prior to its use by
reduction of Fe(CN)63
(
418 = 1012
M1cm1)
(44).
MethodsStopped-flow kinetic measurements were carried out
using an SX-17MV Stopped-Flow from Applied Photophysics coupled with a
1-cm-long mixing cell. Briefly, Mn(III) porphyrins (0.8 to 6 µM)
in acid phosphate solution were mixed in a 1:1 ratio with
ONOO in 10-fold or greater excess, dissolved in a known
concentration of NaOH. The reaction was monitored by the change in absorbance
in the Soret band of the porphyrin, and the plots were fitted to a simple
exponential function. All experiments were carried out at 37 °C. The pH
was measured at the outlet of the stopped flow.
-Radiolysis experiments were carried out with a 137Cs
source (Radiation Machinery Corporation Parsippany, NJ). The dose rate (9.8
gray/min) was determined using the Fricke dosimeter (1 mM
FeSO4 in 0.8 N H2SO4) based on
G(FeIII) = 15.6 and
302(FeIII)
= 2200 M1cm1.
Pulse radiolysis experiments were carried out with a 5-MeV Varian 7715
linear accelerator (0.21.5 µs electron pulses, 200 mA current). All
measurements were made at room temperature in a 1-cm spectrosil cell using
three light passes (optical path length 3.1 cm). A 150-watt xenon-mercury lamp
was used as the light source. The detection system included a Bausch &
Lomb grating monochromator model D330/D331 Mk.11 and a Hamamatsu R920
photomultiplier. The signal was transferred through a Sony/Tektronix 390AD
programmable digitizer to a micro PDP-I 1/24 computer, which operated the
whole pulse radiolysis system.
Generation of
The
radical was generated upon irradiation of N2O-saturated (
25
mM) aqueous solutions containing 0.5 M carbonate at pH
8.5 via the reactions shown below in Equations
2,
3,
4,
5 (the species radiation yields
are given in parentheses in Equation
2).
 | (Eq. 2) |
 | (Eq. 3) |
 | (Eq. 4) |
 | (Eq. 5) |
The pulse intensity was set to produce between 2 and 4 µM
,
and manganese porphyrin concentration ranged between 15 and 60
µM.
Ionization of Axial Water of Mn(III) PorphyrinsSolutions
containing 10 µM Mn(III) porphyrin and 1 M
K2CO3 at pH
13 were mixed in a 1:1 ratio with HCl
solutions of varying concentrations to yield pH 7.513.2. The mixture
was made directly in the spectrophotometer cell using a RX2000 rapid mixing
accessory from Applied Photophysics. Spectra were recorded from 350 to 600 nm
at each pH in a Cary 50 spectrophotometer. Spectral data were analyzed using
Microcal Origin software. MnIIITSPP was dissolved in 0.1
M K2CO3, because the complex precipitates in
more concentrated solutions.
 |
RESULTS
|
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Reaction of Mn(III) Porphyrins with PeroxynitriteThe
reaction of Mn(III) porphyrins with excess of peroxynitrite was studied by
stopped flow. The observed pseudo-first order rate constants were plotted
versus peroxynitrite concentration at each pH to obtain the second
order constant (kox), which increases upon increasing the
pH. Fig. 1 shows plots of
kox versus pH for four representative complexes.
Given that the pKa of ONOOH is 6.6 ± 0.1
(46,
47), our results demonstrate,
as reported previously for several porphyrin complexes
(13), that Mn(III) porphyrins
react faster with ONOO than with
ONOOH.2
 | (Eq. 6) |
 | (Eq. 7) |
 | (Eq. 8) |

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FIG. 1. The rate constants for the reaction of peroxynitrite with Mn(III)
porphyrins as a function of pH in 0.1 M phosphate buffer and 37
°C. Complexes used were MnIIITE-2-PyP (squares),
MnIIITnBu-2-PyP (inverted triangles),
MnIIITM-4-PyP (circles), and MnIIITSPP
(triangles).
|
|
Hence, the effective second-order rate constant of the reaction of Mn(III)
porphyrin with peroxynitrite (kox) is pH-dependent and is
given by Equation 9, where
k6 and k7 are the rate constants of
the reactions shown in Equations
6 and
7, respectively.
 | (Eq. 9) |
Table II summarizes the values
of k6 and pKa obtained for
all ten complexes. The pH profiles of all cationic complexes fit
Equation 9 assuming that
k7 was very small or zero. In the case of
MnIIITSPP, a value of k7 < 105
M1s1 can be put
forward whereas MnIIITCPP did not display any significant variation
in kox in the pH range from 6.4 to 7.5. It is important to
remember that a value of 105
M1s1 is at the low
end of the rate constants measurable by the method used herein. The
pKa values of ONOOH obtained in the case of all
cationic complexes fall below the literature value of 6.6 although it is
higher for the anionic MnIIITSPP
(Table II). This variation can
be explained if the derived pKa values belong to
the outer-sphere complex between ONOO and Mn(III) porphyrin,
and the value deviates from 6.6 because of the relative stabilization of
ONOO by the electric charge on the Mn(III) porphyrin.
Ionization of Axial Water
MoleculesFig.
2A displays the spectral changes experienced by
MnIIITM-2-PyP upon the change in pH from 8.8 to 12.8. No isosbestic
point was detectable in the region from 300 to 600 nm, implying that more than
one equilibrium is involved. Panel B shows the spectrophotometric
titration curve at 454 nm, which is the
max for this
complex at acidic and neutral pH. The plot displays a minimum and two
inflection points, which is consistent with two ionization steps
(pKa1 and pKa2)
shown below in Equations 10 and
11, respectively.
 | (Eq. 10) |
 | (Eq. 11) |

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FIG. 2. Spectrophotometric titration of 5.3 µM
MnIIITM-2-PyP. A, spectra at pH values 8.8, 9.5, 9.8,
10.1, 10.3, 10.6, 11.0, 11.2, 11.4, and 12.8 (some spectra have been omitted
for clarity) at room temperature. The arrows indicate the direction
of change as pH increases from 8.8 to 12.8. B, absorbance at 454 nm
versus pH (error bars are smaller than the
symbols). The solid line represents the best fit to a three
species model (Equation 12) with
pKa1 = 10.5 and pKa2
= 11.4. C, spectra in the regions of 326, 399, 450, 558, and 580 nm
were closely examined and lack isosbestic points. As an example, the titration
curve at 399 nm is shown.
|
|
The spectral data were fitted to Equation
12, shown below, where H2A+ represents
H2O-MnIIIP+, HA represents
OH-MnIIIP, and A represents O =
MnIIIP.
 | (Eq. 12) |
Spectral data at ten significant wavelengths were fitted simultaneously to
four variable parameters: two wavelength-dependent, namely AbsHA,
AbsA, and two wavelength-independent, namely
Ka1 and Ka2. The parameter
AbsH2A+ was obtained from the mean
experimental value at the lowest pH. MnIIITCPP and
MnIIITSPP showed a simpler behavior within the pH range studied,
which was consistent with only one ionization equilibrium; nevertheless,
reported data suggest another pKa below 14
(48). The results are
summarized in Table III.
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TABLE III Axial water pKa1 and pKa2 obtained by fitting the
spectrophotometric titration data to
Equation 12
Titration of 5 µM porphyrin solutions in 0.5 M carbonate buffer in
the range of 7.7 < pH < 13.3 at room temperature.
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Spectral Changes upon Oxidation and Reduction of Mn(III)
PorphyrinsOxidation and reduction of Mn(III) porphyrin were
carried out by
-radiolysis to assess the spectral changes at
500650 nm. O = Mn(IV) porphyrin was produced via the reaction shown in
Equation 13 in
N2O-saturated solutions containing 0.5 M carbonate at pH
10.5.
 | (Eq. 13) |
Mn(II) porphyrin was generated through the reaction shown in
Equation 14, shown below, in
N2O-saturated solutions containing 0.1 M 2-propanol and
50 mM phosphate at pH 7 as described previously
(49).
 | (Eq. 14) |
Difference spectra were calculated as oxidized minus reduced complex. The
difference spectra are exemplified in Fig.
3 for MnIIITM-2-PyP. In all cases the changes in
absorbance associated with the Mn(III) to Mn(IV) transition is about four
times larger than that for the Mn(II) to Mn(III) transition.
Kinetics of the Oxidation of Mn(III) Porphyrins by
The
reaction of 24 µM
with 1560 µM Mn(III) porphyrin was studied at pH
8.513 by pulse radiolysis. The reaction could not be studied at pH <
8.5, because the rate of carbonate oxidation by ·OH (Equations
4 and
5) decrease substantially with
the decrease in the pH, i.e.
pKa(HCO3/CO32)
= 10.2. The reaction was followed at 570575 nm for
MnIIIT-(alkyl)-2(3,4)-PyP5+ and at 595 nm for
MnIIITCPP3 and
MnIIITSPP3. The changes in the
absorbance obeyed first-order kinetics, and kobs was
linearly dependent on the porphyrin concentration
(Fig. 4) and increased upon
increasing the pH as shown in Fig.
5 for four representative complexes. The pH profiles of
k13 were fitted to
Equation 15, below, and the
results are presented in Table
IV.
 | (Eq. 15) |

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FIG. 5. The effect of pH on k13 for four representative
complexes: MnIIITM-4-PyP (squares),
MnIIITM-2-PyP (circles), MnIIITSPP
(triangles), and MnIIITCPP (inverted
triangles). The increment of k13 with pH
correlates with deprotonation of water molecules axially coordinated to the
manganese. Solid lines represent the best fits to
Equation 15. All experiments
were carried out in N2O-saturated solutions containing 0.5
M carbonate buffer.
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|
Oxidation of Mn(II) Porphyrins by
The
reaction of 24 µM
with 1645 µM Mn(II) porphyrin, shown below in
Equation 16, was studied at pH
10.4 by pulse radiolysis.
 | (Eq. 16) |
The reaction was followed at 572 or 600 nm, and k16 was
obtained from the linear dependence of kobs as a function
of [Mn(II) porphyrin] (Table
IV). We assumed that k16 obtained at pH 10.4
is similar to that at neutral pH, as neither
nor Mn(II) porphyrin undergoes acid-base equilibria in this pH range.
 |
DISCUSSION
|
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Reaction of Mn(III) Porphyrins with PeroxynitriteMn(III)
porphyrin reactivity reflects the influence of the substituents on the
porphyrin ring, which is apparent as linear free energy relationships between
different physical-chemical properties of both the free ligand and their metal
complexes (2,
5). Hence,
k6 correlates with E
(Mn(III)/Mn(II)) as
shown in Fig. 6A even
though the reaction shown in Equation
6 does not involve the couple Mn(III)/Mn(II). In fact,
k6 also correlates with the
pKa of the pyrrolic nitrogens of the porphyrin
ring and with the catalytic rate constant of
dismutation in the presence of these complexes (not shown). The reactivity of
the porphyrins toward ONOO thus reflects an overall effect
of the porphyrin ligand on the metal center. This points to an inner-sphere
mechanism where the rate-limiting step is the coordination of
ONOO to the metal, and its rate depends on the resonance and
inductive effects exerted by the porphyrin on the manganese ion.

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FIG. 6. Correlations between k6 (panel A) and log
Ka1 (panel B) versus the redox
potential of the Mn(III)/Mn(II) couple. The numbers indicate the
complexes as follows: 1, MnTCPP; 2, MnTSPP; 3,
MnTM-3-PyP; 4, MnTM-4-PyP; 5, MnTM-2-PyP; 6,
MnTE-2-PyP; 7, MnTnPr-2-PyP; 8, MnTnBu-2-PyP; 9,
MnTnHex-2-PyP; 10, MnTnOct-2-PyP. The linear fit only considers the
first six points in each plot.
|
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The correlation holds as long as the N-alkyl substituents on the
pyridinium groups remain small. For substituents longer than ethyl,
k6 diminishes and remains constant at around (1.3 ±
0.1) x 107
M1s1 in going from
n-propyl to n-octyl. Apparently, the favorable inductive
effect of the ligand (as evidenced from the increase in E
) is
countered by other effects that may involve the decrease in dielectric
constant of the manganese environment. A similar pattern has been found in the
dismutation of superoxide catalyzed by manganese porphyrins
(39).
Ionization of Axial WaterThe
pKa1 of the axial water also show a linear
dependence with E
as apparent in
Fig. 6B. As is the
case with k6, the trend deviates from linearity with
N-alkylpyridinium substituents longer than ethyl. Steric hindrance
cannot be invoked in the acid-base equilibrium of the water protons, because
the dissociation is a unimolecular process that would be influenced by the
local dielectric constant. The water protons are less acidic than expected
from the linear relationship, and this is consistent with charge separation in
the deprotonation process being less favorable in a lower dielectric
environment.
Transmission of Inductive EffectThe inductive effect of the
porphyrin substituents is more noticeable with properties closely related to
the ligand itself. In Table V,
we present the slope of several properties plotted versus E
,
and it can be seen that the slope diminishes as the observed property belongs
to a part of the molecule farther from the meso position. Thus,
pKa(pyrrolic) has the highest value, followed by
kcat and k6 related to the manganese,
and then pKa1, belonging to the axial water
molecules, that perceives the inductive effect through four chemical
bonds.
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TABLE V Slopes of the linear free energy relationship between the listed
properties and the E of the Mn(III)/Mn(II)
couple
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Hydrophobic Effect with Longer Alkyl SubstituentsWe have
seen that N-alkyl-2-pyridinium substituents longer than ethyl caused
a deviation in the linear correlations with E
. This effect is
evident for both k6 and
pKa1(axial) and is similar to what was observed
previously (39) for
kcat for
dismutation. This led us to suppose that the deviation from linearity should
vanish when relating properties that experience the same effect.
Fig. 7 shows the relationships
among the three parameters, all of which display good linear correlations.
This strongly suggests that the effect seen is not related to steric hindrance
(that should not be observable through pKa1) but arises
solely from the decrease in the dielectric constant around the manganese.

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FIG. 7. Correlations among log k6,
pKa1(axial), and log kcat
(
dismutation). The values of kcat were taken from Ref.
39.
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Oxidation of Mn(III) Porphyrins by
The
rate constants of the reaction shown in
Equation 13 differ minimally for
all MnIIIT(alkyl)PyP at low pH levels without any noticeable trend
as is apparent from Table IV,
i.e. k13 = (24) x 108
M1s1.
MnIIITSPP and MnIIITCPP react faster with values of 5.2
x 108 and 1.2 x 109
M1s1, respectively.
If the reaction were by an inner-sphere mechanism, the rate constants should
follow a trend similar to the oxidation by ONOO, correlating
with thermodynamic parameters of the complex and the ligand that reflect the
electronic effect of the substituents. Because this was not the case, the
reaction shown in Equation 13
should follow an outer-sphere mechanism. In this case the rate constants
should correlate with the redox potentials of the Mn(IV)/Mn(III) couple,
according to the Marcus theory. The redox potentials have been determined to
be very similar for some of the complexes at pH 14
(48), with values of 0.382 V
for MnTSPP, 0.388 V for MnTCPP, and 0.406 V for MnTM-2-PyP. Data are scarce at
neutral or slightly alkaline pH, and the values available by
spectroelectrochemistry for MnTM-4-PyP and MnTM-2-PyP at neutral pH are close
to 1.0 V versus normal hydrogen electrode
(41,
50). Values for MnTCPP can
only be extrapolated from results at pH > 10
(48). In any case, all values,
either measured or extrapolated, cluster around 1 V versus normal
hydrogen electrode. The values obtained for the
reaction, if an outer-sphere mechanism is assumed, imply that the redox
potentials of the complexes should differ by less than 0.1 V.
The increment of k13 with pH
(Fig. 5) correlates with
deprotonation of axial water molecules. Our spectrophotometric titration
results show that all MnIIIT(alkyl)-2(3,4)-PyP have two ionization
equilibria in the pH range from 9 to 13.2, whereas MnIIITSPP and
MnIIITCPP have higher values. The pH profiles of
k13 can be fitted using the
pKa values obtained through spectrophotometric
titration and presented in Table
III, but this yields ambiguous results. All
MnIIIT(alkyl)-2(3,4)-PyP fit well to a single ionization
equilibrium model. This can be explained by considering the species
H2O-MnIIIP+, HO-MnIIIP, and O =
MnIIIP as before. If HO-MnIIIP has a
reactivity similar to the average between
H2O-MnIIIP+ and O =
MnIIIP only one
pKaapp will be visible from the
reactivity trend as a function of pH, and that
pKaapp will be roughly halfway between
pKa1 and pKa2. A
comparison between the pKa values presented in
Tables III and
IV shows that this is the case
for all MnIIIT(alkyl)-2-PyP, within 0.3 pH units.
Our data are not sufficient to estimate the acid-base behavior of the
reactivity with MnIIITSPP and MnIIITCPP, but the
limiting value at high pH can be estimated at 1.2 x 109 and
2.8 x 109
M1s1, respectively.
Considering the pKa1 obtained by
spectrophotometric titration, further extrapolation is not possible at this
stage.
Oxidation of Mn(II) Porphyrins by
The
rate constant of the reaction shown in
Equation 16 determined herein by
pulse radiolysis falls in the range of 1 to 5 x 109
M1s1, which makes
Mn(II) porphyrins good scavengers of
with reactivities well above those of biologically relevant targets
(25).
Peroxynitrite,
and
Their ScavengingThe results presented herein support the idea of
manganese porphyrins as valuable tools in the scavenging of peroxynitrite and
the species derived from it, such as
.
On kinetic grounds, the similar reactivities of all MnT(alkyl)-2-PyP make them
promising candidates to directly intercept ONOO and suppress
most of the
that might be formed. The reactions of Mn(III) porphyrins with both
and ONOO form O = Mn(IV) porphyrin that reacts
preferentially with endogenous antioxidants such as ascorbate and urate
(13) and less efficiently with
seemingly more critical targets such as thiols and amino acids.
Because the Mn(III) porphyrins can be reduced by biological reductants,
such as ascorbate, the Mn(II) porphyrins could mediate the oxidation of such
expendable and regeneratable reductants by
and in so doing protect critical cellular targets. The reactivity of Mn(II)
porphyrins with ONOO remains virtually unexplored, but
preliminary data suggest that the reaction is fast and does not produce
secondary oxidant
radicals.3 Finally,
the different correlations found here represent an important inferential tool
in estimating Mn(III) porphyrin reactivity toward superoxide and peroxynitrite
and embody relevant information for drug design directed to oxygen and
nitrogen species-mediated tissue injury.
 |
FOOTNOTES
|
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* This work was supported in part by grants from the International Centre for
Genetic Engineering and Biotechnology (Italy), The Howard Hughes Medical
Institute (to R. R.), and by Comisión Sectorial de Investigación
Científica (CSIC, Uruguay) (to G. F. S.). The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
Received travel grants from Programa de Desarrollo de Ciencias
Básicas and CSIC to visit the Hebrew University, Jerusalem, Israel. 
¶ Received a young researcher scholarship from CSIC. 
** Recipient of Christopher Reeve Paralysis Foundation Grant BA1-0103-1 and
supported in part by Aeolus/Incara. 

Recipient of National Institutes of Health Grant R01DK59868 and supported
in part by the Amyotrophic Lateral Schlerosis Association. 
||||
International Research Scholar of the Howard Hughes Medical Institute. To whom
correspondence should be addressed. Tel.: 5982-9249561; Fax: 5982-9249563;
E-mail:
rradi{at}fmed.edu.uy.
1 The abbreviations used are: MnIIITCPP, manganese(III)
meso-tetrakis(4-carboxylatophenyl)porphyrin;
MnIIITM-2-PyP,
manganese-(III)meso-tetrakis((N-methyl)pyridinium-2-yl)porphyrin;
MnIIITM-4-PyP,
manganese(III)meso-tetrakis((N-methyl)pyridinium-4-yl)porphyrin;
MnIIITSPP,
manganese(III)meso-tetrakis(4-sulfonatophenyl)-porphyrin;
MnIIITE-2-PyP,
manganese(III)meso-tetrakis((N-ethyl)pyridinium-2-yl)porphyrin;
MnIIITnPr-2-PyP,
manganese(III)meso-tetrakis((N-n-propyl)pyridinium-2-yl)porphyrin;
MnIIITnBu-2-PyP,
manganese(III)meso-tetrakis((N-n-butyl)pyridinium-2-yl)porphyrin;
MnIIITnHex-2-PyP,
manganese(III)meso-tetrakis((N-n-hexyl)pyridinium-2-yl)porphyrin;
MnIIITnOct-2-PyP,
manganese(III)meso-tetrakis((N-n-octyl)pyridinium-2-yl)porphyrin;
MnIIITM-3-PyP,
manganese(III)meso-tetrakis((N-methyl)pyridinium-3-yl)porphyrin. 
2 The electric charge of the complexes in the equations only considers the
formal charge of the metal ion and the atoms directly bound to it, thus
Mn(III) porphyrin is MnIIIP+, Mn(II) porphyrin is
MnIIP, and O = Mn(IV) porphyrin is O = MnIVP. 
3 G. Ferrer-Sueta, I. Batini
-Haberle, and R. Radi, manuscript in
preparation. 
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