Reactions of Manganese Porphyrins with Peroxynitrite and Carbonate Radical Anion*

Gerardo Ferrer-Sueta {ddagger} §, Darío Vitturi {ddagger} , Ines Batinic-Haberle || **, Irwin Fridovich || {ddagger}{ddagger}, Sara Goldstein §§, Gidon Czapski §§ and Rafael Radi ¶¶ ||||

From the {ddagger}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
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 M–1 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 M–1s1 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 M–1s1 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 7.5–13.2 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 E1/2 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
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 M–1s1 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 {approx} 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 ({epsilon}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, E1/2 (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
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—Mn(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 ({epsilon}418 = 1012 M–1cm1) (44).


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TABLE I
Structure, abbreviation, and electric charge at pH 7 of the Mn(III) complexes used in this work

 

Methods—Stopped-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.

{gamma}-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 {epsilon}302(FeIII) = 2200 M–1cm1.

Pulse radiolysis experiments were carried out with a 5-MeV Varian 7715 linear accelerator (0.2–1.5 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) Porphyrins—Solutions 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 7.5–13.2. 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
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reaction of Mn(III) Porphyrins with Peroxynitrite—The 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 M–1s1 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 M–1s1 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.


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TABLE II
Second order rate constants for the reaction shown in Equation 6 and apparent pKa values for peroxynitrite obtained from fitting the data of kox to Equation 9, at 37 ± 0.1 °C

ND, not detected.

 

Ionization of Axial Water Molecules—Fig. 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 {lambda}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 {approx}5 µM porphyrin solutions in 0.5 M carbonate buffer in the range of 7.7 < pH < 13.3 at room temperature.

 

Spectral Changes upon Oxidation and Reduction of Mn(III) Porphyrins—Oxidation and reduction of Mn(III) porphyrin were carried out by {gamma}-radiolysis to assess the spectral changes at 500–650 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.



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FIG. 3.
Difference spectra obtained upon oxidation of MnIIITM-2-PyP by (solid line) and reduction of MnIIITM-2-PyP by 2-hydroxypropyl radical (dashed line) using {gamma}-radiolysis at room temperature. Solid line, (Mn(IV) – Mn(III)) was obtained upon irradiation (58.8 gray) of 50 µM MnIIITM-2-PyP in N2O-saturated solutions containing 0.5 M carbonate at pH 10.5. Dashed line, (Mn(III) – Mn(II)) was obtained upon irradiation (58.8 gray) of 50 µM MnIIITM-2-PyP in N2O-saturated solutions containing 0.1 M phosphate and 0.1 M 2-propanol at pH 7.

 

Kinetics of the Oxidation of Mn(III) Porphyrins by The reaction of 2–4 µM with 15–60 µM Mn(III) porphyrin was studied at pH 8.5–13 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 570–575 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. 4.
The observed first order rate constant for the oxidation of MnIIITM-2-PyP by at pH 8.9 (filled squares) and pH 12 (open squares). All experiments were carried out at room temperature in N2O-saturated solutions containing 0.5 M carbonate and = 2–4 µM.

 


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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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TABLE IV
Rate constants for the oxidation of manganese porphyins by at room temperature

 

Oxidation of Mn(II) Porphyrins by —The reaction of 2–4 µM with 16–45 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
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reaction of Mn(III) Porphyrins with Peroxynitrite—Mn(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 E1/2(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.

 

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 M–1s1 in going from n-propyl to n-octyl. Apparently, the favorable inductive effect of the ligand (as evidenced from the increase in E1/2) 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 Water—The pKa1 of the axial water also show a linear dependence with E1/2 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 Effect—The 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 E1/2, 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 E1/2 of the Mn(III)/Mn(II) couple

 

Hydrophobic Effect with Longer Alkyl Substituents—We have seen that N-alkyl-2-pyridinium substituents longer than ethyl caused a deviation in the linear correlations with E1/2. 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.

 

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 = (2–4) x 108 M–1s1. MnIIITSPP and MnIIITCPP react faster with values of 5.2 x 108 and 1.2 x 109 M–1s1, 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 M–1s1, 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 M–1s1, which makes Mn(II) porphyrins good scavengers of with reactivities well above those of biologically relevant targets (25).

Peroxynitrite, and Their Scavenging—The 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
 
* 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. Back

§ Received travel grants from Programa de Desarrollo de Ciencias Básicas and CSIC to visit the Hebrew University, Jerusalem, Israel. Back

Received a young researcher scholarship from CSIC. Back

** Recipient of Christopher Reeve Paralysis Foundation Grant BA1-0103-1 and supported in part by Aeolus/Incara. Back

{ddagger}{ddagger} Recipient of National Institutes of Health Grant R01DK59868 and supported in part by the Amyotrophic Lateral Schlerosis Association. Back

|||| 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. Back

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. Back

3 G. Ferrer-Sueta, I. Batinic-Haberle, and R. Radi, manuscript in preparation. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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