Electrostatic Contribution in the Catalysis of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> Dismutation by Superoxide Dismutase Mimics

MnIIITE-2-PyP5+ VERSUS MnIIIBr8T-2-PyP+*

Ivan Spasojevic'Dagger §, Ines Batinic'-HaberleDagger , Júlio S. Rebouças||, Ynara Marina Idemori||, and Irwin FridovichDagger

From the Dagger  Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 and the  Departamento de Química, ICEx, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, 31270-901, Brazil

Received for publication, November 6, 2002, and in revised form, December 2, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnIIITE-2-PyP5+) is a potent superoxide dismutase (SOD) mimic in vitro and was beneficial in rodent models of oxidative stress pathologies. Its high activity has been ascribed to both the favorable redox potential of its metal center and to the electrostatic facilitation assured by the four positive charges encircling the metal center. Its comparison with the non-alkylated, singly charged analogue Mn(III) beta-octabromo meso-tetrakis(2-pyridyl)porphyrin (MnIIIBr8T-2-PyP+) enabled us to evaluate the electrostatic contribution to the catalysis of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> dismutation. Both compounds exhibit nearly identical metal-centered redox potential for MnIII/MnII redox couple: +228 mV for MnIIITE-2-PyP5+ and +219 mV versus NHE for MnIIIBr8T-2-PyP+. The eight electron-withdrawing beta pyrrolic bromines contribute equally to the redox properties of the parent MnIIIT-2-PyP+ as do four quaternized cationic meso ortho pyridyl nitrogens. However, the SOD-like activity of the highly charged MnIIITE-2-PyP5+ is >100-fold higher (log kcat = 7.76) than that of the singly charged MnIIIBr8T-2-PyP+ (log kcat = 5.63). The kinetic salt effect showed that the catalytic rate constants of the MnIIITE-2-PyP5+ and of its methyl analogue, MnIIITM-2-PyP5+, are exactly 5-fold more sensitive to ionic strength than is the kcat of MnIIIBr8T-2-PyP+, which parallels the charge ratio of these compounds. Interestingly, only a small effect of ionic strength on the rate constant was found in the case of penta-charged para (MnIIITM-4-PyP5+) and meta isomers (MnIIITM-3-PyP5+), indicating that the placement of the positive charges in the close proximity of the metal center (ortho position) is essential for the electrostatic facilitation of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> dismutation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The thermodynamic (1, 2-8) and electrostatic effects (9-16) in the catalysis of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> dismutation by superoxide dismutases have been extensively studied. The redox potential of all superoxide dismutases was found to be similar, independently of the type of the metal in the active site. It is around midway (+360 mV versus NHE)1 (17) between the potential for the oxidation (-160 mV versus NHE) and for the reduction of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> (+890 mV versus NHE). Thus it allows equal driving force (hence kox = kred = ~2 × 109 M-1 s-1) for both half-reactions of the catalytic cycles (Reactions 1 and 2) (18-20). For Escherichia coli Fe-SOD,
<UP>Mn<SUP>III</SUP>SOD</UP>+<UP>O</UP><SUB><UP>2</UP></SUB><SUP><UP>&cjs1138;</UP></SUP><UP> ⇔ Mn<SUP>II</SUP>SOD</UP>+<UP>O<SUB>2</SUB>,</UP>k<SUB><UP>ox</UP></SUB>

<UP><SC>Reaction</SC> 1</UP>

<UP>Mn<SUP>II</SUP>SOD</UP>+<UP>O</UP><SUB><UP>2</UP></SUB><SUP><UP>&cjs1138;</UP></SUP>+<UP>2H<SUP>+</SUP> ⇔ Mn<SUP>III</SUP>SOD</UP>+<UP>H<SUB>2</SUB>O<SUB>2</SUB>,</UP>k<SUB><UP>red</UP></SUB>

<UP><SC>Reaction</SC> 2</UP>
the E1/2 is +223 mV versus NHE at pH 7.4, and for Mn-SOD from the Bacillus stearothermophilus and E. coli the E1/2 were +260 and +310 mV versus NHE at pH 7 (1, 21, 22). However, when manganese was replaced by iron in the active site of Mn-SOD the enzymatic activity was lost (18), which has been attributed to the decrease of the redox potential below that required for the oxidation of superoxide ion (3, 18). Such metal ion specificity (2) has been recently explained by the higher affinity of Fe3+ than Mn3+ for hydroxide (6, 8).

The crystal structures of different SODs reveal a highly conserved electrostatic "funnel" (13, 14, 16) that is believed to guide the negatively charged superoxide toward the active site of the enzyme. In the past there have been considerable efforts to evaluate the extent of electrostatic facilitation, but a major difficulty lies in the inability to specifically modify the positively charged residues without affecting the structural integrity of the active site (9).

The Mn(III) porphyrin, MnIIITE-2-PyP5+ (AEOL-10113) has been shown (23-27) to possess high SOD-like activity in vitro with log kcat = 7.76. The compound has further proven effective in protection of SOD-deficient E. coli (26) and in stroke (28, 29), spinal cord injury (30, 31), diabetes (32, 33), sickle cell disease (34), and radiation/cancer (35-38) rodent models of oxidative stress injuries. Much like SOD (1, 9, 21, 22) its high catalytic potency has been ascribed both to the favorable redox properties of the metal center and to the effect of the positively charged ortho-N-ethylpyridyl nitrogens that provide electrostatic facilitation for the approach of the negatively charged superoxide (23).

The MnIIITE-2-PyP5+ (Scheme I) exists as a mixture of rotational isomers (39). Expectations that the four positive charges in alpha alpha alpha alpha isomer will guide the superoxide anion toward the metal center in a cooperative fashion making it the most powerful SOD mimics among the isomers, proved to be groundless; all four isomers were found to be of equal catalytic potency.


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Scheme I.   Structures of the MnIIITE(M)-2-PyP5+, MnIIITM-3-PyP5+, MnIIITM-4-PyP5+, and MnIIIBr8T-2-PyP+.

Recently, the synthesis and characterization of the beta -brominated non-N-alkylated analogue of MnIIIT-2-PyP+ has been reported (40). The electron-withdrawing effect of the beta -pyrrolic bromines on the redox properties of the metal center of the porphyrins (50-70 mV/bromine) has been previously established (41-47). The effect of eight beta -pyrrolic bromines was expected to be similar in magnitude to the effect of the four quaternized pyridyl nitrogens on the redox properties of the starting unsubstituted MnIIIT-2-PyP+ porphyrin molecule.

Herein, we show that the redox properties of the brominated non-N-alkylated and the N-alkylated Mn(III) ortho-pyridylporphyrins are indeed nearly identical, allowing us to evaluate the electrostatic contribution in the catalysis of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> dismutation. Similar studies would be hard to conduct on the enzymes themselves. Thus our findings give us the unique opportunity to understand the relative importance of thermodynamics and kinetics in the O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> dismutation by the superoxide dismutases (18-20).

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials

General-- Xanthine and ferricytochrome c were from Sigma, and NaCl, KOH, KH2PO4, methanol, and EDTA were from Mallinckrodt. Xanthine oxidase was prepared by R. Wiley and was supplied by K. V. Rajagopalan (48). Catalase was from Roche Molecular Biochemicals, ultrapure argon was from National Welders Supply Co., and Tris buffer (ultrapure) was from ICN Biomedicals, Inc.

Mn(III) Porphyrins-- The H2T-2-PyP+ and MnIIITM-3(4)-PyP5+ were obtained from MidCentury Chemicals (Chicago, IL). MnIIITE(M)-2-PyP5+ (26, 27) and MnIIIBr8T-2-PyP+ (40) were prepared as previously described. The molar absorptivities of the Soret bands of MnTM-2-PyP5+ (log epsilon 453.4 = 5.11), MnTM-3-PyP5+ (log epsilon 459.8 = 5.14), MnTM-4-PyP5+ (log epsilon 462.2 = 5.11), and MnTE-2-PyP5+ (log epsilon 454 = 5.14) (26, 27) all in water and of MnIIIBr8T-2-PyP+ (log epsilon  482 = 4.66) in acetonitrile were used for quantitation. Due to the low water solubility, a 2 mM stock solution of MnIIIBr8T-2-PyP+ in methanol was used throughout this study.

Methods

Electrochemistry-- Measurements were performed on a CH Instruments Model 600 voltammetric analyzer. A three-electrode system was utilized with a glassy carbon (3 mm) or gold (2 mm) button working electrode (Bioanalytical Systems), a Ag/AgCl reference, and a platinum wire as auxiliary electrode. Due to the low water solubility of the MnIIIBr8T-2-PyP+, electrochemical studies of both compounds were performed in 9/1 (v/v) methanol/aqueous solutions as previously reported (49). The 9/1 (v/v) methanol/aqueous solutions contained 0.05 M Tris, pH 7.8, 0.1 M NaCl, and 0.3 mM metalloporphyrin. Tris buffer was used instead of phosphate buffer, because the latter precipitates in methanol. The potentials were standardized against potassium ferrocyanide/ferricyanide (51) and MnIIITE-2-PyP5+. The redox potential of the MnIII/MnIV redox couple, which was previously found to be proton-dependent (52) was determined at pH 12.3. The scan rates were 0.01-10 V/s. The E1/2 values for MnII/MnIII and MnIII/MnIV redox couples obtained in 9/1 (v/v) methanol/aqueous solutions were extrapolated to aqueous medium values as previously described (49).

Catalysis of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> Dismutation-- We have previously shown that the convenient cytochrome c assay gives the same catalytic rate constants as does pulse radiolysis in the case of MnIIITE-2-PyP5+, {MnIIIBVDME}2, {MnIIIBV2-}2, and MnIICl2 (49), and it was therefore utilized in this study. The xanthine/xanthine oxidase reaction was the source of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, and ferricytochrome c was used as the indicating scavenger for O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> (53). The reduction of cytochrome c was followed at 550 nm. Assays were conducted at 25 ± 1 °C, in 0.05 M phosphate buffer, pH 7.8, 0.1 mM EDTA, 10 µM cytochrome c, 40 µM xanthine, with or without 15 µg/ml catalase. Aqueous stock solutions of MnIIITE(M)-2-PyP5+ and MnTM-3(4)-PyP5+ and the methanolic stock solution of MnIIIBr8T-2-PyP+ were diluted into the assay mixture. Rate constants for the reaction of metalloporphyrins with O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> were based upon competition with 10 µM cytochrome c as described elsewhere (49). The kcyt c = 2.6 × 105 M-1 s-1 obtained under the same experimental conditions (pH 7.8, 21 °C, 0.05 M phosphate buffer, 0.1 mM EDTA) (50) was used to calculate kcat. The O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> was produced at the rate of 1.2 µM per minute. Any possible interference through inhibition of the xanthine/xanthine oxidase reaction by the test compounds was examined by following the rate of urate accumulation at 295 nm in the absence of cytochrome c. No reoxidation of ferrocytochrome c by metalloporphyrins was observed. No effect of catalase was detected implying sufficient stability of the compounds toward H2O2. We have previously determined the rate constants for the degradation of MnIIITE-2-PyP5+ and of MnIIITM-2-PyP5+ by H2O2 to be 1.3 M-1 s-1 and for MnIIITM-3-PyP5+ and MnIIITM-4-PyP5+ 4.9 and 4.6 M-1 s-1, respectively. In this work we found that the MnIIIBr8T-2-PyP+ proved to be at least two orders of magnitude more stable.

Kinetic Salt Effect-- The dependence of the catalytic rate constant for the O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> dismutation upon ionic strength was determined in 0.05 M phosphate buffer, pH 7.8, with NaCl ranging from 0 to 0.4 M.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Electrochemistry

The MnII/MnIII Redox Couple-- Reversible cyclic voltammograms of the MnII/MnIII redox were obtained for both compounds, MnIIITE-2-PyP5+ and MnIIIBr8T-2-PyP+, at scan rates of 0.1 V/s (Fig. 1). Thus it was possible to determine the half-wave potentials, E1/2, given in Table I. The two compounds have almost identical MnII/MnIII metal-centered E1/2 values at pH 7.8, as predicted from the number and the nature of the electron-withdrawing substituents on the meso positions of the porphyrin ring (41-47). Moreover, their voltammetric behavior in terms of electrochemical reversibility (peak-to-peak potential separation, Delta Epp (Fig. 2A), and current response to a change in scan rate (Fig. 2C)) as well as the chemical reversibility (ratio between the reduction and oxidation peak currents (Fig. 2B)) is strikingly similar, which leaves us to believe that the difference in the reactivity toward superoxide is indeed due to the difference in the overall positive charge (electrostatic attraction of superoxide anion) and not due to a difference in the rates of electron transfer (electronic and structural differences).


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Fig. 1.   A, cyclic voltammograms of MnIIITE-2-PyP5+ (Mn-2E5+) and MnIIIBr8T-2-PyP+ (Mn-Br8+) obtained at pH 7.8 in 9/1 (v/v) methanol/aqueous solution, 0.05 M Tris buffer, 0.1 M NaCl, scan rate 0.1 V/s. B, cyclic voltammograms of MnIIITE-2-PyP5+ (Mn-2E5+) and MnIIIBr8T-2-PyP+ (Mn-Br8+) obtained at pH 12.3 in 9/1 (v/v) methanol/aqueous solution, 0.05 M Tris buffer, 0.1 M NaCl, scan rate 0.1 V/s. Solid line, first cycle; dashed lines, subsequent cycles.

                              
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Table I
The E1/2 for the MnII/MnIII and MnIII/MnIV redox couples and kcat for the O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> dismutation by MnIIITE-2-PyP5+ and MnIIIBr8T-2-PyP+


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Fig. 2.   Electrochemistry of MnIIITE-2-PyP5+ (Mn-2E5+) and MnIIIBr8T-2-PyP+ (Mn-Br8+) at pH 7.8 in 9/1 (v/v) methanol/aqueous solution, 0.05 M Tris buffer, 0.1 M NaCl. A, peak-to-peak potential separation as a function of log (scan rate). B, ratio of the reduction and oxidation peak currents as a function of log (scan rate). C, the reduction and oxidation peak currents as a function of the square root of the scan rate.

The MnIII/MnIV Redox Couple-- Redox properties of the Mn sites were further explored by studying the MnIII/MnIV redox couple. Because the hydroxo-MnIII and oxo-MnIV species are involved (52), this redox process is accessible only in basic solution and is proton-dependent. At pH 12.3, reversible cyclic voltammograms were obtained in the case of both compounds, MnIIITE-2-PyP5+ and MnIIIBr8T-2-PyP+, with essentially equal MnIII/MnIV redox couple potentials of +381 mV and +372 mV versus NHE, respectively (Fig. 1B and Table I). The MnIII/MnIV and MnII/MnIII redox processes are independent as demonstrated on Fig. 1B (dashed traces), whereas reversible voltammetric waves were obtained even when the cycling was done only in a narrow potential range around the E1/2 of the corresponding redox couple.

Because a deprotonation of the axially ligated water on Mn(III) porphyrins occurs at pH 12.3, the MnII/MnIII redox potential shifts negatively (52). Therefore, we ascribe the 145-mV difference in the shift between the two redox couples (Fig. 1B and Table I) to a difference in the pKa,ax values of their axially ligated water. We have previously found that E1/2 reflects the electron density of the porphyrin ring and the metal center in such a way that there is a linear relationship between the pKa of the pyrrolic nitrogen protons of the porphyrin ligand and the metal-centered E1/2 for a series of differently substituted manganese porphyrins (26). We have further found that the axial ligation also is influenced by the electron density of the metal center; in the case of ortho, meta, and para MnTM-2-PyP5+ more positive E1/2 correlates with lower pKa,ax (52). However, in the case of a series of Mn(III) ortho N-alkylpyridylporphyrins (alkyl = methyl through octyl) we saw that hydrophobic effects may reverse the trend (54). With more hydrophobic members of the series, despite a more positive E1/2, the creation of charge is hindered resulting in higher pKa values of the pyrrolic nitrogens of the parent ligands (54). In the present work at pH 12.3, the hydrophobic MnIIIBr8T-2-PyP+ with presumably higher pKa,ax (thus resisting deprotonation), exhibits a lower shift of the MnII/MnIII couple than highly hydrophilic MnIIITE-2-PyP5+. In the case of the MnIII/MnIV couple, which is also proton-dependent (52), there was practically no difference in E1/2 between the two compounds, which is in line with findings reported by us (24, 49-52) and others (55-61) that MnIII/MnIV redox potential is fairly insensitive to the porphyrin structure.

Catalysis of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> Dismutation-- The catalytic rate constants were calculated from the linear plots of v0/vi - 1 versus concentration obtained from the spectrophotometric cytochrome c assay measurements, as described elsewhere (49). The kcat values determined in 0.05 M phosphate buffer, pH 7.8, are given in Table I. The MnIIIBr8T-2-PyP+ is >100-fold less efficient a catalyst than MnIIITE-2-PyP5+.

Kinetic Salt Effect-- The effect of the ionic strength (µ) on the catalytic rate constant was assessed using Equation 1, which is based on the Debye-Huckel relation (62) for the effect of the ionic strength of the solution on the activity coefficient of an ion,


<UP>log</UP>k=<UP>log</UP>k<SUB><UP>ref</UP></SUB>+<UP>2</UP>Az<SUB><UP>A</UP></SUB>z<SUB><UP>B</UP></SUB><UP> &mgr;</UP><SUP><BINOM><NU><UP>1</UP></NU><DE><UP>2</UP></DE></BINOM></SUP><UP>/</UP>(<UP>1</UP>+<UP>&mgr;</UP><SUP><BINOM><NU><UP>1</UP></NU><DE><UP>2</UP></DE></BINOM></SUP>) (Eq. 1)
where k is the rate constant at any given ionic strength, and kref is the rate constant at m = 0. The A is a collection of physical constants with a value of 0.509, and zA and zB are the charges of the reacting species. The equation predicts a linear plot of log k versus µ1/2/(1 + µ1/2). Equation 1 assumes a coefficient of 1.0 (beta ai) for µ1/2 in the denominator, i.e. the distance of the closest approach, ai to be 3 Å, and beta  is a physical constant, 0.33 × 10-10 m-1. It is doubtful whether great significance can be attributed to the ai, thus to the product zAzB (39), especially so in the light of the bulkiness, high charge, and solvation shell of the metalloporphyrins. Accounting for the mono- and diprotonated phosphates as the major species at pH 7.8 (pKa = 7.2), and the concentration of the NaCl, the ionic strength was calculated using Equation 2, where mi is the molality and zi the charge of the given ion,
<UP>&mgr;</UP>=<UP>½</UP><LIM><OP>∑</OP></LIM><UP>m</UP><SUB><UP>i</UP></SUB>z<SUP><UP>2</UP></SUP><SUB><UP>i</UP></SUB> (Eq. 2)
Linear plots of log kcat versus µ1/2/(1 + µ1/2) (Equation 1) are presented in Fig. 3. The slopes of the plots are -6.96 (MnIIITE-2-PyP5+), -6.93 (MnIIITM-2-PyP5+), -2.57 (MnIIITM-3-PyP5+), -2.60 (MnIIITM-4-PyP5+), and -1.41 (MnIIIBr8T-2-PyP+). The intercepts present kref and are 9.71 (MnIIITM-2-PyP5+), 9.68 (MnIIITE-2-PyP5+), 7.25 (MnIIITM-3-PyP5+ and MnIIITM-4-PyP5+), and 6.01 (MnIIIBr8T-2-PyP+). As expected (54), when the reactants are ions of opposite charges, the higher the ionic strength of the solution the lower the rate constants. Methyl and ethyl ortho isomers, MnIIIE(M)-2-PyP5+ behave equally with respect to kinetic salt effect. The ratio of slopes for MnIIITE(M)-2-PyP5+ and MnIIIBr8T-2-PyP+ was found to be 4.9, which equals the ratio of their charges. The kinetic salt effect of meta and para isomers, MnIIITM-3-PyP5+ and MnIIITM-4-PyP5+, was also assessed. Only a small effect of the positive charges was observed when they were placed peripherally with respect to metal center in para and meta isomers, MnIIITM-3 (4)-PyP5+ (Fig. 3).


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Fig. 3.   Log kcat versus1/2(1 + µ1/2) for MnIIITM-2-PyP5+, MnIIITM-3-PyP5+, MnIIITM-4-PyP5+, MnIIITE-2-PyP5+, and MnIIIBr8T-2-PyP+ obtained in 0.05 M phosphate buffer, pH 7.8, 0-0.4 M NaCl. Slopes are given in parentheses. The intercepts present kref and are 9.71 (MnIIITM-2-PyP5+), 9.68 (MnIIITE-2-PyP5+), 7.25 (MnIIITM-3-PyP5+ and MnIIITM-4-PyP5+), and 6.01 (MnIIIBr8T-2-PyP+).


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Log kcat versus E1/2-- To design a potent, low molecular weight SOD mimic we aimed at approaching the E1/2 of the enzyme and affording electrostatic facilitation. We established a relationship between the log kcat and the E1/2 for the MnII/MnIII redox couple for a series of Mn(III) porphyrins. An increase in E1/2 of 120 mV caused a 10-fold increase in kcat (26), which is in agreement with the Marcus equation for an outer-sphere electron transfer (63, 64). At potentials that are negative with respect to the midway potential, a Mn+3 oxidation state is stabilized, and the reduction of metalloporphyrin becomes rate-limiting. The preliminary data indicate that, similar to the SODs, when E1/2 approaches the midway potential for O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> reduction and oxidation of ~ +360 mV versus NHE, the rate constants for the reduction (Reaction 1 and oxidation of manganese porphyrins (Reaction 2) by O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> become similar (65). Thus the reduction of MnIIITM-4-PyP5+ at E1/2 = +62 mV versus NHE, is 1000-fold slower than its oxidation (65), whereas the reduction of MnIIITE-2-PyP5+, at E1/2 = +228 mV versus NHE, is only <= 4-fold slower than its oxidation. Thus in a preliminary pulse radiolysis study the rate constant for the reduction of MnIIITE-2-PyP5+ by O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> was found to be 2.5 × 107 M-1 s-1, whereas it was 8.2 × 107 M-1 s-1 for its oxidation.2 As the potential increases further, the Mn+2 oxidation state becomes stabilized and the oxidation of metalloporphyrin becomes rate-limiting (22, 37, 66). Mn(II) porphyrins can efficiently catalyze dismutation of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>. The MnIIBr8TM-4-PyP4+ and MnIICl5TE-2-PyP4+ do so with log kcat = 8.34 (41) and 8.41 (66), respectively. However, both compounds suffer from insufficient metal/ligand stability to be used in vivo as SOD mimics (41, 66).

Based on the log kcat versus E1/2 relationship, the compound MnIIITE-2-PyP5+ was chosen as the most promising one for in vivo testing. It dismutes O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> with high catalytic rate constant of 5.8 × 107 M-1s-1 at E1/2 of +228 mV versus NHE, and it affords electrostatic facilitation in the catalysis while retaining metal/ligand stability.

Deviation from the Plot Log kcat versus E1/2-- Although dismutation appears to be an outer-sphere electron transfer, electrostatic, steric, and solvation effects, which are not accounted for by the Marcus equation, do play a role. When one or more of these effects predominate over E1/2, deviation from Marcus plot occurs. The highest degree of deviation was observed with the series of Mn(III) ortho N-alkylpyridyl porphyrins and with the singly charged MnIIIBr8T-2-PyP+. In the case of the former porphyrins, an interplay of steric and solvation effects results in a "V" shape dependence of log kcat upon E1/2 (54). Thus, as the alkyl chains lengthen from methyl to n-octyl accompanied by an increase in hydrophobicity, the E1/2 steadily increases. Yet, from methyl to n-butyl the kcat decreases, whereas it increases from n-butyl to n-octyl. In the case of singly charged MnIIIBr8T-2-PyP+, as discussed below, the deviation from the Marcus plot originates from the lack of electrostatic facilitation.

Electrostatics versus Redox Potential in the Catalysis of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> Dismutation by SOD Mimics-- Like MnIIITE-2-PyP5+, the singly charged MnIIIBr8T-2-PyP+ is a derivative of the same parent compound, MnIIIT-2-PyP+. When eight electron-withdrawing bromines are placed on the beta -pyrrolic positions of MnIIIT-2-PyP+, they cause a positive shift in E1/2 of the MnII/MnIII redox couple of 499 mV (Table I and Fig. 1). Our finding is in agreement with available literature on the effect of beta -bromination on the E1/2 of metalloporphyrins (41-47). An almost identical increase in E1/2 (508 mV) was achieved by placing four quaternized ortho-pyridyls in the meso positions of the MnIIIT-2-PyP+. Thus the E1/2 of MnIIITE-2-PyP5+ and MnIIIBr8T-2-PyP+ are +228 mV and +219 mV versus NHE, respectively (Table I and Fig. 1). Even though the E1/2 values for the MnII/MnIII redox couple responsible for O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> dismutation are essentially identical, MnIIITE-2-PyP5+ was found to be >100-fold more efficient catalyst of O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> dismutation. The corresponding log kcat are 7.76 and 5.63 for MnIIITE-2-PyP5+ and MnIIIBr8T-2-PyP+, respectively (Table I). Thus we conclude that the effect of such a magnitude originates entirely from electrostatic facilitation of the O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> dismutation.

As an additional support for the importance of the electrostatics in the O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> dismutation, the kinetic salt effect was assessed to see whether a 5-fold charge difference causes a 5-fold higher susceptibility of the catalytic rate constant of MnIIITE-2-PyP5+ to the ionic strength when compared with the kcat of the singly charged MnIIIBr8T-2-PyP+. The linear plots of log kcat versus µ1/2/(1 + µ1/2) (Equation 1), obtained for both compounds (Fig. 3), show that the ratio of slopes (4.9) equals the ratio of charges, which clearly establishes the impact that electrostatics has on the SOD-like catalysis. The same effects of the ionic strength on kcat were observed in the case of ethyl and methyl analogues, MnIIITE(M)-2-PyP5+ (Fig. 3). The meta and para isomers, MnIIITM-3 (4)-PyP5+, were also studied to determine the importance of the location of the positive charges. Very little salt effect was observed when the charges are further away from the metal center; thus the meta and para isomers behaved much as did the MnIIIBr8T-2-PyP+ (Fig. 3). Therefore, close proximity of the positive charges is essential for O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> guidance. These data strongly point to the metal center as the site of electron transfer and suggest that the dismutation might not be truly outer-sphere in that there might be some bonding interactions during the encounter of the reactants.

    ACKNOWLEDGEMENTS

We are thankful for the financial support provided by Christopher Reeve Paralysis Foundation and by Aeolus/Incara Pharmaceuticals, Research Triangle Park, NC. J. S. R. and Y. M. I. gratefully acknowledge the financial support from The Brazilian Research Council (CNPq) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (Brazil).

    FOOTNOTES

* This work was supported in part by Grant BA1-0103-1 from the Christopher Reeve Paralysis Foundation and by Aeolus/Incara Pharmaceuticals, Research Triangle Park, NC.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 919-684-2101; Fax: 919-684-8885; E-mail: ivan@chem.duke.edu.

|| Supported by The Brazilian Research Council and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (Brazil).

Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M211346200

2 J. Grodkowski, I. Batinic-Haberle, P. Neta, and I. Fridovich, unpublished data.

    ABBREVIATIONS

The abbreviations used are: NHE, normal hydrogen electrode; SOD, superoxide dismutase; meso, refers to the substituents at the 5, 10, 15, and 20 (meso carbon) position of the porphyrin core; beta , refers to the substituents at beta -pyrrolic carbons; MnIIIT-2-PyP+, Mn(III) 5,10,15,20-tetrakis(2-pyridyl)porphyrin; MnIIITE-2-PyP5+ (Mn-2E5+) (AEOL-10113), manganese(III) 5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin; MnIIITM-2(3, 4)-PyP5+ (Mn-2(3,4)M5+), manganese(III) 5,10,15,20-tetrakis(N-methylpyridinium-2(3,4)-yl)porphyrin, where 2 (AEOL-10112), 3, and 4 refer to ortho, meta, and para isomers, respectively; MnIIIBr8T-2-PyP+ (Mn-Br8+), Mn(III) 2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetrakis(2-pyridyl)porphyrin; MnIIBr8TM-4-PyP4+, Mn(II) 2,3,7,8,12,13,17,18-octabromo-5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin; MnIICl5TE-2-PyP4+, Mn(II) beta -pentachloro-5,10,15,20-tetrakis(N-ethylpyridinium-2-yl)porphyrin; {MnIIIBV2-}2, Mn(III) biliverdin IX; {MnIIIBVDME}2, Mn(III) biliverdin IX dimethyl ester.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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