manganese(iii) complexes with porphyrins and related compounds as catalytic scavengers of superoxide

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Inorganica Chimica Acta 317 (2001) 230–242

Manganese(III) complexes with porphyrins and related compoundsas catalytic scavengers of superoxide

Ivan Spasojevic, Ines Batinic-Haberle *Department of Biochemistry, Duke Uni�ersity Medical Center, Durham, NC 27710, USA

Received 8 November 2000; accepted 16 January 2001

Abstract

Two groups of Mn-based catalytic antioxidants are described in terms of their catalytic activities and electrochemical properties.In the first group, manganese porphyrins, phthalocyanine, and porphyrazine employ the Mn(III)/Mn(II) couple in the catalysisof O2

�− dismutation (disproportionation). The catalytic rate constant is dependent upon the metal-centered redox potential, asshown previously for water-soluble Mn porphyrins. The limitation of this simple relation becomes obvious with compounds ofhigh redox potential (+2 metal oxidation state is stabilized) which exhibit a weak metal/ligand binding; although of highsuperoxide dismutase (SOD)-like activity, the compounds are not stable under physiological conditions. The second generation ofthe potent O2

�− scavengers are manganese complexes with biliverdin IX and its derivatives which have an RO− functionality asa fifth coordination to the metal center in a dimeric structure. Such a coordination pattern stabilizes the +4 oxidation state ofthe manganese so that the Mn(III)/Mn(IV) redox (E1/2= � +0.46 V vs. NHE) becomes coupled to the O2

�− dismutation. Moreimportantly, despite operating at a high positive metal-centered redox potential and having the+3 oxidation state as the restingstate of the metal center, metallobiliverdins still retain a high ligand affinity in solution. Independently of their charge (two neutraland the other two negatively charged) metallobiliverdins studied are of similar SOD-like activity comparable to the efficacy ofhighly charged manganese(III) ortho N-alkylpyridylporphyrins. These most potent in vitro SOD-like Mn porphyrins are alsoreactive towards peroxynitrite, nitric oxide, hydrogen peroxide and oxygen. Since the fifth coordination site of the metal centeris occupied no reactivity of the manganese(III) biliverdin IX dimethyl ester towards NO� and H2O2 is observed. Thus,manganese(III) porphyrins and manganese(III) biliverdins are expected to differ with regards to their tissue localization and to thetype and the concentration of reactive oxygen species they would encounter in biological systems. Comparative kinetic andthermodynamic studies of these catalytic antioxidants would help us understand not only the prevalent mode of their in vivobiological action but the mechanism of oxidative stress injuries as well. © 2001 Elsevier Science B.V. All rights reserved.

Keywords: Manganese complexes; Porphyrin complexes; Superoxide; Superoxide dismutase mimics

1. Introduction

Reactive oxygen and nitrogen species have been im-plicated in numerous pathological processes includinginflammation, ischemia/reperfusion, hemorrhagic shock,autoimmune diseases, neurological disorders, radiationinjuries, carcinogenesis and senescense [1–14]. Syntheticand natural compounds capable of reacting catalyti-cally or stoichiometrically with one or more of these

reactive species have been developed and proven effec-tive in different in vivo models of oxidative stress[15–39]. Our laboratory has been engaged in designingsuperoxide dismutase (SOD)-mimics that are eithermetalloporphyrins or their derivatives [27–30,32,–39].The metal center was chosen to be manganese due to itsbiocompatibility while the cyclic structure of the ligandoffers a high metal/ligand stability. The first generationof these compounds is the water-soluble manganeseporphyrins. Those described in this work are presentedin Scheme 1. For these manganese complexes wehave established a linear free-energy relationship be-tween the log kcat for dismutation (disproportionation)of O2

�− and the metal-centered redox potential

* Corresponding author. Tel.: +1-919-6842101 fax: +1-919-6848885.

E-mail addresses: [email protected] (I. Spasojevic),[email protected] (I. Batinic-Haberle).

0020-1693/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved.

PII: S 0 0 20 -1693 (01 )00365 -6

I. Spasoje�ic, I. Batinic-Haberle / Inorganica Chimica Acta 317 (2001) 230–242 231

Scheme 1. Manganese(III) porphyrins, Mn(III) phthalocyanine and Mn(III) porphyrazines.

[27]. When this relationship is combined with effectsof metalloporphyrins on the growth of SOD-deficientEscherichia coli, we conclude that manganese(III)porphyrins of metal-centered redox potential � +50mV versus NHE would have kcat high enough to be con-sidered potentially efficient antioxidants.

The most potent antioxidants in vitro [27,28] and invivo [27,36–38], that are still of satisfactory metal/lig-and stability, the ortho isomers of manganese(III) N-alkylpyridylporphyrins, are also reactive towardsbiologically active oxygen or nitrogen species other thansuperoxide anion. Their reactivities towards nitric oxide[39], peroxynitrite [32], hydrogen peroxide [27,28] and

oxygen [39,40] have been reported as well. Thus theobserved protection in different in vivo models of oxida-tive stress [27,28,36–38] may originate from the reac-tions with any of those species and would invariablydepend upon the concentration of the reactive speciesand upon their tissue localization. Another possiblemode of action might be a ‘free’ manganese releaseduring the reduction of the metal center (weakening ofthe metal/ligand bond), independently of the nature ofthe reducing species that may be either O2

�−, ascorbicacid, glutathione, NO�, etc. In vitro and in vivo antiox-idant effect of manganese has already been documented[41–45].

I. Spasoje�ic, I. Batinic-Haberle / Inorganica Chimica Acta 317 (2001) 230–242232

Scheme 2. Biliverdin IX and its derivatives and their Mn(III) complexes.

The cationic metalloporphyrins of high positivecharge tend to interact with nucleic acids [46–51].However, steric hindrance may partially diminish thateffect; the para isomer, Mn(III)TM-4-PyP5+, which ismore planar associates much more with DNA andRNA then the ortho isomer, Mn(III)TM-2-PyP5+ [28].In addition, depending upon the specific function ex-pected from such compounds, the association with nu-cleic acids may be either beneficial or undesirable.

Since highly efficient metalloporphyrin-based cata-lytic scavengers of O2

�− are of high positive chargewhich is expected to govern their tissue localization andinfluence their reactivity, we have extended our studiesto the characterization of water-insoluble singly-charged Mn(III) porphyrins, Mn(III) porphyrazines,and Mn(III) phthalocyanine shown in Scheme 1.

We are currently developing a series of second gener-ation of the manganese(III) complexes which differ inlipophilicity, metal/ligand stability and reactivity to-wards small reactive biological molecules when com-pared to manganese(III) porphyrins [33]. In suchcompounds the manganese is ligated to biliverdin IX orto its derivatives forming a dimeric structure (Scheme

2). Both biliverdin and bilirubin have been recentlyshown to exhibit neuroprotective effects and their pos-sible antioxidant activity in vivo has been postulated[52–56]. It has also been suggested [57], based upon animpressive heme degradation activity of brain uponwhich iron, biliverdin, and CO are formed, that hemeoxygenase system, consisting of constitutive heme oxy-genase-2 and oxidative stress-inducible protein hemeoxygenase-1, has other functions aside from hemedegradation. The possible involvement of heme oxyge-nase in the mechanisms for mutual regulatory interac-tions between CO- and NO�-generating systems hasbeen addressed [57]. We have demonstrated that invitro metal-free biliverdin and bilirubin ligands arereactive neither towards superoxide nor hydrogen per-oxide despite their rather favorable redox chemistry[33,58]. While bilirubin ligand has no appreciableaffinity towards formation of an Mn complex [59–62],biliverdin, in contrast, readily forms Mn(III) biliverdincomplex with high SOD-like activity [33]. We can thenspeculate that biliverdin liberated by the action of hemeoxygenase may form Mn biliverdin complex in situ andas such express an oxidative stress relieving activity.


Table 1UV–Vis and electrochemical characterization of biliverdin IX and its derivatives

Ligand �max (nm)E1/2 (V vs. NHE) a �×104 (M−1 cm−1)Ligand oxidation

660Biliverdin IX 12.9+0.59Biliverdin IX dimethyl ester +0.59 666 11.8 b

Biliverdin IX ditaurate 655+0.57 10.8632+0.59 13.7Mesobiliverdin IX dimethyl ester

a E1/2 values relate to the one-electron, one-proton redox and are determined in 9/1 (v/v) methanol+aqueous solutions, 0.05 M tris buffer, pH*7.9, 0.1 M NaCl, and extrapolated [33] to aqueous medium values (overall uncertainty estimated to be �10 mV).

b Data from Ref. [33].

In this work, the study has been extended to biliv-erdin IX itself and its derivatives and their mangan-ese(III) complexes [63]. Understanding their propertiesand their in vivo action might help us to understand theprotective function of heme-oxygenase [57,64–66].

2. Experimental

2.1. Materials

N,N-Dimethylethanolamine (99.5%), manganese(II)acetate tetrahydrate (99.99%), N,N-dimethylformamide(99.8% anhydrous), manganese(II) chloride tetrahy-drate (99.99%) were from Aldrich. Glacial acetic acid(�99.7%) was from EM Science. Acetone (99.7%),EDTA and methanol (99.9%) were from Mallinkrodt.Chelex 100, 200–400 mesh, sodium form was fromBIO-RAD. Pyridine-2,3-dicarbonitrile was from TCIAmerica, and pyridine-3,4-dicarbonitrile (98+%) wasfrom Lancaster. Tris (ultra pure) was from ICNBiomedicals, Inc. Biliverdin IX dihydrochloride(�80%) and N,N-bis(salicylidene)ethylenediamine(H2salen) (99+%) were from Fluka. Biliverdin IXdimethyl ester was used as obtained from FrontierScientific, Logan, Utah. Biliverdin IX ditaurate andmesobiliverdin IX dimethyl ester were a generous giftfrom J. C. Bommer. Mn(III)TPP+, Mn(III)T(PFP)P+,Mn(III)T-4-PyP+, M(III)T-2-PyP+, Mn(III)TM-3(4)-PyP5+, Mn(III)T(TMA)P5+, Mn(III)TCPP3−,Mn(III)T(2,6-Cl2-3-SO3-P)P3−, Mn(III)T(2,6-F2-3-SO3-P)P3−, Mn(III)T(TFTMA)P5+ were used as obtainedfrom Mid-Century Chemicals, Chicago, IL.Mn(III)salen+ was prepared as previously described[33]. Mn(III)PcCl was obtained from Aldrich.Mn(III)TM(E)-2-PyP5+ (available from Mid-CenturyChemicals, Chicago, IL), Mn(III)BMT-2-PyP3+ andMn(III)TrM-2-PyP4+ were prepared as described al-ready [27]. Mn(III)TE-4-PyP5+ was prepared in thesame way as its ortho analogue. Tetrakis(2,3-pyridino)porphyrazine, H2T-2,3-PyPz was prepared asreported previously [67,68]. The metallation wasachieved with manganese(II) acetate at 1:10 ligand to

metal ratio in refluxing glacial acetic acid where 1%pyridine was added to improve the ligand solubility[69]. The Mn complex formation was evident from thedisappearance of the ligand absorption at 646 nm(DMF) and appearance of a band at 674 nm (DMF).The glacial acetic acid vas evaporated under vacuo, theresidue washed several times with water and dried invacuo at room temperature. The concentration of themetal complex was determined using the reported mo-lar absorptivity of the ligand, log �643=4.82 (DMF)[70]. H2T-3,4-PyPz and its Mn(III) complex was pre-pared in the same manner [67,68] as H2T-2,3-PyPz andits Mn(III) complex. H2T-3,4-PyP+ has the absorptionat 668 nm (DMF), while its Mn complex has a band at707 nm (glacial acetic acid). Since the position ofpyridyl nitrogens was found to affect the position ofSoret bands of ortho, meta and para isomers ofMn(III)TMPyP5+ but insignificantly their correspond-ing molar absorptivities [28], we used the molar absorp-tivity of H2T-2,3-PyPz to calculate the concentration ofMn(III)T-3,4-PyP+. The alkylation of the pyridyl nitro-gens of Mn(III)T-2,3-PyPz+ and Mn(III)T-3,4-PyPz+

was attempted but so far unsuccessfully. That is mostprobably due to the destabilization of the porphyrazineby further decreasing already electron-deficient por-phyrazine ring.

The manganese(III) complex with biliverdin IXdimethyl ester was prepared as described previously[33]. The manganese complexes with biliverdin IX,mesobiliverdin IX dimethyl ester and biliverdin IX di-taurate were prepared in 9/1 (v/v) methanol + aqueoussolution, 0.05 M tris buffer, pH* (see Abbreviations)7.9 at 10% excess MnCl2. Upon completion of metalla-tion the solutions of the Mn(III) complexes were usedfor electrochemical and uv/vis characterization and forkcat determination. The concentration of the Mn(III)complexes was calculated using molar absorptivities ofthe ligands given in Table 1.

2.2. Methods

2.2.1. UV–Vis spectroscopyThe UV–Vis measurements were performed on UV-

2501PC Shimadzu spectrophotometer at (25�1)°C.


The molar absorptivities of biliverdin IX and its deriva-tives were determined in methanol and are given inTable 1. The formation of their Mn complexes wasfollowed in 9/1 (v/v) MeOH+H2O, 0.05 M tris buffer,pH* 7.9 at 10% excess metal. The oxidation of{Mn(III)BVDME}2 and its parent ligand was followedspectrophotometrically in oxygen- and argon-saturated9/1 (v/v) MeOH+H2O, 0.05 M tris buffer, pH* 7.9.

The UV–Vis spectra of 6–60 �M Mn(III)salen+

(�235=3.7×104 M−1 cm−1, �279=1.70×104 M−1

cm−1, �397=4.60×103 M−1 cm−1) [33] in the presenceand absence of 100-fold excess of EDTA and in thepresence of Chelex were made in 0.05 M phosphatebuffer, pH 7.8.

2.2.2. ElectrochemistryCyclic voltametry was performed using CH Instru-

ments model 600 voltammetric analyzer with a 3 mmdiameter glassy carbon button working electrode (Bio-analytical Systems), Ag � AgCl reference electrode (3 MNaCl, Bioanalytical Systems) and a Pt wire (0.5 mm) asauxiliary electrode [33]. Typically the concentration ofthe manganese complexes and biliverdin ligands was�0.5 mM. Compounds were dissolved in either 9/1(v/v) methanol+aqueous solution, 0.05 M tris buffer,pH* 7.9, 0.1 M NaCl, or 8.5/0.5/1 (v/v/v) methanol+

Table 3Metal-centered redox potentials and catalytic rate constants for dis-mutation of O2

�− by manganese(III) porphyrins, manganese(III)phthalocyanine and manganese(III) tetrakis(2,3-pyridino)porphyr-azine

E1/2 (V vs. NHE) log kcataCompound

Mn(III)/Mn(II)

−0.28 bMn(III)T-2-PyP+ 4.29Mn(III)T-4-PyP+ 4.53−0.20 b

−0.19 cMn(II)TCPP3− 4.56−0.27 bMn(III)TPP+ 4.83

Mn(III)T(PFP)P+ −0.12 b 5.00Mn(III)T(TMA)P5+ 5.11−0.10 c

−0.01 cMn(III)T(2,6-F2-3-SO3-P)P3− 5.51+0.09 cMn(III)T(2,6-Cl2-3-SO3-P)P3− 6.00

6.08+0.06 cMn(III)T(TFTMA)P5+

+0.05 cMn(III)BM-2-PyP3+ 6.52Mn(III)TM-4-PyP5+ +0.06 c 6.58

+0.05 cMn(III)TM-3-PyP5+ 6.616.63+0.12 cMn(III)TrM-2-PyP4+

+0.07 dMn(III)TE-4-PyP5+ 6.86Mn(III)TE-2-PyP5+ +0.23 c 7.76Mn(III)TM-2-PyP5+ +0.22 c 7.79

−0.03 eMn(III)Pc+ 5.816.46Mn(III)T-2,3-PyPz+ +0.09 f

+0.20 fMn(III)T-3,4-PyPz+ g

a The kcat was determined by cytochrome c assay at (25�1)°C withuncertainty estimated to be �20%. The data for water-solubleporphyrins bearing 3+ to 5+ and 3− charges, except Mn(III)TE-4-PyP5+ (this work) are from Ref. [27].

b E1/2 values were determined in 9/1 (v/v) methanol+aqueoussolutions, 0.05 M tris buffer, pH* 7.9, 0.1 M NaCl; given are theextrapolated [33] E1/2 values to aqueous medium values.

c E1/2 values were determined in 0.05 M phosphate buffer, pH 7.8,0.1 M NaCl, the data are taken from Ref. [27] except for Mn(III)TE-4-PyP5+.

d E1/2 was determined in 0.05 M phosphate buffer, pH 7.8, 0.1 MNaCl.

e E1/2 was determined in 8.5/0.5/1 (v/v/v) methanol+aqueous+pyridine solution, 0.05 M tris buffer, pH* 7.9, 0.1 M NaCl andextrapolated to aqueous medium value. Pyridine was used to increasethe solubility of Mn(III)PcCl.

f E1/2 was determined in 9/1 (v/v) DMF+aqueous solutions, 0.05M tris buffer, pH* 7.9, 0.1 M NaCl and extrapolated to aqueousmedium value.

g We were not able to determine the SOD-like activity, presumablydue to the aggregation. E1/2 values were determined with uncertaintyof �3 mV.

Table 2Metal-centered redox potentials and catalytic rate constants for O2

�−dismutation for manganese(III) biliverdin IX and related compounds

E1/2 (V vs. log kcatbE1/2 (V vs.Compound

NHE) aNHE) a

Mn(III)/Mn(IV) Mn(III)/Mn(II)

7.40−0.30+0.46{Mn(III)BV2−}2

−0.23 c+0.45 c 7.70 c{Mn(III)-BVDME}2

+0.44{Mn(III)- −0.26 7.36MBVDME}2

7.40+0.47{Mn(III)- −0.26BVDT2−}2

a E1/2 values were determined in 9/1 (v/v) methanol + aqueoussolutions, 0.05 M tris buffer, pH* 7.9, 0.1 M NaCl, and are extrapo-lated to aqueous medium values [33]. The values refer to the follow-ing redox reactions: 1/2{Mn(III)BV2−}2�1/2{Mn(IV)BV−}2+e−,1/2{Mn(III)BV2−}2+H++e−�Mn(II)HBV2−. Voltammogramsobtained have peak-to-peak separation of 59 mV or higher whichmeans that redox processes at two metal centers occur independently[33]. No redox activity of ligands was observed in the region wheremetal-centered redox activity was found (Table 1). E1/2 values weredetermined with uncertainty of �3 mV.

b Catalytic rate constants were determined by cytochrome c assayat (25�1)°C with uncertainty of �20%; the constants are calculatedper manganese concentration.

c Data from Ref. [33].

aqueous+pyridine solution, 0.05 M tris buffer, pH*7.9, 0.1 M NaCl, or 9/1 (v/v) DMF+aqueous solution,0.1 M NaCl, 0.05 M tris buffer, as noted in Tables 1–3.Cyclic voltametry of Mn(III)TE-4-PyP5+ was done in0.05 M phosphate buffer, pH 7.8, 0.1 M NaCl. Thecalibration performed using either K3Fe(CN)6, fer-rocenemethanol, or Mn(III)TM-2-PyP5+ all gave samevalues for redox potentials in V versus NHE. There-fore, the cyclic voltammogram of Mn(III)TM-2-PyP5+,obtained under same experimental conditions as thevoltammogram of other manganese complexes, was


used for calibration. Often Mn(III)TM-2-PyP5+ wasadded as internal standard directly into solutions beingstudied.

2.2.3. Catalysis of O2�− dismutation

The catalysis of O2�− dismutation was followed by

xanthine/xanthine oxidase/cytochrome c assay as de-scribed previously [27,33,71,72]. We [27,33] and others[73,74] have shown that both the cytochrome c assayand the pulse radiolysis are equally reliable for evalua-tion of the catalytic properties of Mn(III) porphyrinsand Mn(III) biliverdins. The 0.5 mM methanolic stocksolutions of singly-charged and uncharged Mn(III) por-phyrins and Mn(III) biliverdins (concentration meantper Mn) were diluted into 0.05 M phosphate bufferassay solution. In the case of Mn(III)Pc+ andMn(III)T-2,3-PyPz+ the 0.5 mM solutions, used todetermine E1/2, were utilized as stock solutions for kcat

determination. The solvent system is indicated in Table3.

3. Results

The molar absorptivities of biliverdin ligands weredetermined in the concentration range from 10−6 to10−5 M where adherence to Beer–Lambert law wasobeyed. The data are given in Table 1.

At 1:1 metal to ligand ratio the metallation of biliv-erdins goes to completion indicating a high metal/lig-and stability. It has been shown that no dechelation ofMn(III) biliverdin IX dimethyl ester happens even at900-fold excess EDTA [33]. All Mn(III) biliverdins

exhibit the same UV–Vis characteristics as shown forMn(III) biliverdin IX in Fig. 1 where its metallation in9/1 (v/v) methanol+aqueous solution, 0.05 M trisbuffer, pH* 7.9 was followed. Therefore, the Mn(III)complex of biliverdin IX, {Mn(III)BV2−}2, and of itsderivatives were likely to be of the same dimeric struc-ture that we established previously [33] for the man-ganese(III) biliverdin IX dimethyl ester (Scheme 2). AllMn(III) complexes as well as their respective ligands[this work and refs 63 and 75] are sensitive to oxidation.In the presence of oxygen, initially a slow metal-cen-tered oxidation takes place. The spectral change thataccompanies that process shown in Fig. 2(A) for{Mn(III)BVDME}2 is the same as observed previously[33] upon the spectroelectrochemical oxidation of thecomplex at +500 mV versus Ag � AgCl (Fig. 2(B)) andascribed to Mn(III) to Mn(IV) oxidation [33]. It alsoresembles the spectral change obtained when{Mn(III)BVDME}2 was oxidized by trichloro-methylperoxyl radical, produced upon irradiation inaerated methanol solution in the presence of CCl4, orby chloroperoxybenzoic acid [33]. Once oxidized at themetal site the manganese(IV) complex undergoes fur-ther change observed through the appearance of a bandat �640 nm. We ascribe that change to the oxidationof the ligand by Mn(IV) (Fig. 2(A)). This assignment issupported by the appearance of the same band in theuv/vis spectrum of the ligand when it is left in anoxygen-saturated solution (Fig. 2(C)). Under argon nometal oxidation is observed and the spectrum of{Mn(III)BVDME}2 undergoes a slow change (Fig.2(D)) resembling the oxidation of the ligand itself underoxygen-saturated conditions. We conclude that the lig-and oxidation in {Mn(III)BVDME}2 complex couldhave happened only at the expense of an internalMn(III)-to-Mn(II) redox. Either ligand oxidation ormetal reduction leads eventually to the decompositionof the dimeric structure and to the loss of metal.Further work is in progress [63] to study the impact ofthe differences in the structures of biliverdin derivativeson the kinetics of their oxidative degradation and thedegradation of their Mn(III) complexes.

The E1/2 values that correspond to the reversibleoxidation of the ligands are given in Table 1. A pHincrease from 7.9 to 10.0 resulted in a −120 mV shiftof the voltammetric wave of biliverdin IX dimethylester as predicted by Nernst equation for one-protonone-electron redox (−59 mV per pH unit [76]) andshould hold for all other biliverdins studied.

The manganese(III) biliverdins studied have similarmetal-centered redox potential (Table 2). A representa-tive voltammogram of manganese(III) biliverdin IX isshown in Fig. 3. At 1 V/s the voltammetric wavecorresponding to the Mn(III)/Mn(II) redox couple isirreversible. Presumably, the reduced form of the metalcomplex undergoes demetallation on the time scale of

Fig. 1. Formation of 20 �M {Mn(III)BV2−}2 in 9/1 (v/v) methanol+ aqueous solution, pH* 7.9, 0.05 M tris buffer at 10% excess metal.Spectrum 1 was obtained in the absence of metal salt, while spectrum9 was taken 40 min upon the addition of MnCl2.


Fig. 2.

the cyclic voltammetry experiment. However, a partialreversibility was achieved at faster scan rate (10 V s−1)as shown in Fig. 3. We have determined previously [33]that the Mn(III)/Mn(IV) redox of {Mn(III)BVDME}2

is proton-independent while the Mn(III)/Mn(II) redoxis proton-dependent. The same should be true for allMn(III) biliverdins, being of comparable structure asevidenced by essentially identical UV–Vis spectra andredox potentials. The equations related to the redoxcouples of {Mn(III)BV2−}2 are given in the footnote ofTable 2.

The metal-centered redox potentials of Mn(III) por-phyrins, Mn(III) phthalocyanine (Mn(III)Pc+), andMn(III) porphyrazines (Mn(III)T-2,3-PyPz+, Mn(III)-T-3,4-PyPz+) are given in Table 3. The data for thewater-soluble porphyrins are from Ref. [27]. Both orthoMn(III) porphyrin, Mn(III)T-2-PyP+, and its por-phyrazine analogue, Mn(III)T-2,3-PyPz+, have metal-centered redox potentials shifted negatively byapproximately 100 mV relative to the metal-centeredredox potentials of their para analogues, MnT-4-PyP+

and MnT-3,4-PyPz+. This may be understood in termsof a stronger electron-donating effect of an ortho thanof a para(meta) pyridyl nitrogens. Such effect is com-parable in magnitude but opposite in direction to theelectron-withdrawing effect that quaternized pyridyl ni-trogens impose when being in ortho relative to paraposition.

The catalytic rate constants for the dismutation ofO2

�− are given in Tables 2 and 3. The data for thewater-soluble Mn porphyrins were taken from Ref. [27]and for {Mn(III)BVDME}2 from Ref. [33]. All Mn(III)biliverdins, two negatively charged and two neutral, areof similar SOD-like activity, i.e. no significant effect ofcharge is observed. Their enzymatic activity resemblesthe activity of the Mn(III) ortho N-alkylpyridylpor-phyrins (Tables 2 and 3). Due to the high level ofaggregation we were not able to determine the SODactivity of the Mn(III)T-3,4-PyPz+. The singly-chargedMn(III) porphyrins studied are of poor SOD-like activ-ities (Table 3).

Fig. 2. (A) Time-dependent oxidation of 9 �M {Mn(III)BVDME}2 inthe oxygen-saturated 9/1 (v/v) MeOH + H2O solution, 0.05 M trisbuffer, pH* 7.9 (A) (t=1.0, 6.0, 23.3, 75.7 h). (B) Spectroelectro-chemistry of 3 mM {Mn(III)BVDME}2 in 9/1 (v/v) MeOH + H2O,0.05 M tris buffer, pH* 7.9 at applied potential of +500 mV vs.Ag � AgCl within 9 min. (C) Time-dependent oxidation of 22 �MBVDME3− in the oxygen-saturated 9/1 (v/v) MeOH + H2O solu-tion, 0.05 M tris buffer, pH* 7.9, t=1.4 h, 56.8 h, 77.5 h, 96.1 h,125.8 h. (D) Time-dependent oxidation of 9 �M {Mn(III)BVDME}2

in the argon-saturated 9/1 (v/v) MeOH + H2O solution, 0.05 M trisbuffer, pH* 7.9 (t=1.0, 56.5, 99.9, 148.8 h).


Fig. 3. Electrochemistry of {Mn(III)BV2−}2 in 9/1 (v/v) MeOH +H2O, 0.05 M tris buffer, pH* 7.9 (see also Table 2).

shows that for each 120 mV increase in E1/2 there is atenfold increase in kcat, consistent with the Marcusequation [81] for an outer-sphere electron transfer.

The metal-centered redox potential is found furtherto be proportional to the proton dissociation constantsof pyrrolic nitrogens of the metal-free ligands (H3P+�H2P+H+, P=porphyrin) [27]. (pKa and E1/2 bothreflect the porphyrin structure while log kcat describesthe SOD-like activity; thus we refer to the linear free-energy relationship also as a structure–activity relation-ship [92]) The higher the catalytic rate constant, thehigher the metal-centered redox potential, the lower thepKa and consequently the lower the ligand bindingability. Therefore at high positive potentials (� +0.46V vs. NHE) comparable to the potentials of Mn(III)biliverdins, either the Mn(III) porphyrin complexwould be of low stability [34] or the +2 oxidation stateof the Mn would be preferred resulting in an unstableMn(II) complex [29,82]. The SOD-like activity ofMn(III)Cl4TE-2-PyP5+ (log kcat=8.60, E1/2= +0.45V vs. NHE) [34], Mn(II)Cl5TE-2-PyP4+ (log kcat=8.41, E1/2= +0.56 V vs. NHE) [82] and Mn(II)Br8TM-4-PyP4+ (log kcat=8.34, E1/2= +0.48 V vs. NHE) [29]is higher than that of Mn(III)TM(E)-2-PyP5+

(log kcat=7.76(7.79), E1/2= +0.22(0.23) V vs. NHE)[27,34] but at the expense of their decreased metal/lig-and stability.

Herein we have revised a linear free-energy relation-ship by including the Mn(III) porphyrins that bearsingle positive charge and are essentially water-insolu-ble and also Mn(III) phthalocyanine and Mn(III) te-trakis(2,3-pyridino)porphyrazine presented in Scheme1. We showed that they too obey the same Marcusequation for an outer-sphere electron transfer. Obvi-ously, the metal-centered redox potential is a majorfactor affecting SOD-like activity in vitro. However, thecompounds that are of negative charge or bear a singlepositive charge deviate most from the relationship indi-cating that the electrostatics, as well as the overallstructure of the complex, have some impact on thecatalysis.

When compared to porphyrins, the porphyrazineshave a decreased macrocycle electron density, whichresults in several orders of magnitude lower protondissociation constants of pyrrolic nitrogens and in thevery acidic meso nitrogens (pKa= −0.15) [83–87].When additional electron-withdrawing functionalities[84,87,88] are placed on the porphyrazine ring, theligand itself and its metal complex become unstable asevidenced by our inability to N-alkylate Mn(III)T-2,3-PyPz+ and Mn(III)T-3,4-PyPz+.

We have proven [33] that manganese(III) biliverdinIX dimethyl ester is of same dimeric structure (Scheme2) as shown by Balch and collaborators for a similarcompound, manganese(III) octaethylbilindione [89,90].The UV–Vis spectra of Mn complexes with other biliv-erdins are identical to the UV–Vis spectrum of

4. Discussion

The superoxide dismutases [77] catalyze the dismuta-tion of O2

�− at � +300 mV versus NHE regardless ofthe type of the metal center of the enzyme involved[78,79]. This potential is around midway between thepotential where O2

�− is being reduced (+0.87 V vs.NHE) and oxidized (−0.16 V vs. NHE) [80], sinceenzyme oxidizes and reduces O2

�− at identical rates[79]. The most powerful in vitro low-molecular weightwater-soluble SOD mimics, manganese(III)N-methyl(M)- and N-ethyl(E)pyridyl porphyrins,Mn(III)TM(E)-2-PyP5+, operate at similar potentialsof +0.22(0.23) V versus NHE [27] (Eqs. (1) and (2)).

Mn(III)TM(E)-2-PyP5+ +O2�−

�Mn(II)TM(E)-2-PyP4+ +O2 (1)

Mn(II)TM(E)-2-PyP4+ +O2�− +2H+

�Mn(III)TM(E)-2-PyP5+ +H2O2 (2)

In such compounds the oxidation of O2�− (Eq. (1)) is

slower than its reduction and thus is the rate-limitingstep ([27] and refs. therein). Under such conditions it isexpected that the redox potential of a potent metal-loporphyrin-based SOD mimic should be more positivethan that of the SOD enzyme, the optimal value beingwhen rates of both oxidation and reduction of O2

�−become equal. The linear free-energy relationship deter-mined previously for a series of water-soluble mangane-se(III) porphyrins [27], which utilize Mn(III)/Mn(II)couple for O2

�− dismutation (and are in solution in +3resting state) showed that the catalytic rate constant forO2

�− dismutation, i.e. the rate-limiting O2�− oxidation,

is continuously increasing with the metal-centered re-dox potential. That means that in practice, an optimalredox potential, i.e. the highest SOD-like activity, is yetto be achieved. The slope of the linear regression line


{Mn(III)BVDME}2 suggesting that the manganese(III)complexes with biliverdin IX, mesobiliverdin IXdimethyl ester and biliverdin IX ditaurate are alldimeric (Scheme 2). The dimeric structure, where eachmanganese of one biliverdin unit satisfies the 5th coor-dination through binding to the enolic oxygen of an-other biliverdin unit (Scheme 2), governs the differencesin their electrochemistry when compared to the electro-chemistry of manganese(III) porphyrins. In Mn(III)biliverdins the Mn(III)/Mn(II) redox occurs at potentialtoo negative (−0.23 V to −0.3 V vs. NHE) to allowany catalysis of O2

�− dismutation. However it is thestabilization of the manganese in its +4 oxidationstate, whereby the Mn(III)/Mn(IV) couple is at a favor-able redox potential (+0.44 to +0.46 V vs. NHE),that enable these compounds to be effective catalystsfor O2

�− dismutation (Eqs. (3) and (4)). All Mn(III)biliverdins have similar E1/2 of Mn(III)/Mn(IV) couple,and accordingly they all are of similar SOD-like activity(Table 2).

1/2{Mn(III)BV2−}2+O2�− +2H+

�1/2{Mn(IV)BV−}2+H2O2 (3)

1/2{Mn(IV)BV−}2+O2�−�1/2{Mn(III)BV2−}2+O2

(4)

In contrast to Mn(III) porphyrins, the pentacoordi-nate nature of the metal site prevents manganese(III)biliverdins from reacting with NO� and makes theminert towards H2O2 [33]. It is because of this increasedselectivity that Mn(III) biliverdins may be utilized inunderstanding the in vivo mechanism of action of com-pounds that are characterized in vitro by high SOD-likeactivities, but may in vivo react with reactive oxygenspecies other than superoxide. This may ultimately leadto better understanding of the mechanism of oxidativestress in general.

In vitro most effective SOD mimic is Mn(III)TM(E)-2-PyP5+ [27,28,32]. The difference in the reactivity ofthe N-methylated and N-ethylated analogues is in-significant [27,33,34,39]. In Scheme 3 we have summa-rized data so far collected for Mn(III)TM(E)-2-PyP5+.Besides O2

�−, the compound also reacts with a varietyof oxygen and nitrogen species (Scheme 3)[27,28,32,39,91]. When Mn is in its +3 oxidation state,the compound has essentially equal reactivity towardsO2

�− and ONOO− and is inert towards NO�. Whenreduced, in addition to reaction with O2

�−, the com-pound rapidly reacts with NO� [39] and O2 (Scheme 3)[39,91] and could release metal as data on demetallationof Mn(II)TM-4(2)-PyP4+ in aqueous acidic solutions[93–99] would indicate. Mn(II) porphyrin is tenfold lessprone to oxidative degradation by H2O2 than Mn(III)porphyrin [92]. Preliminary study [91] showed that inthe presence of ascorbic acid Mn(II)TE-2-PyP4+ reactswith S-nitrosoglutathione (GSNO), leading to the for-

mation of nitrosylated compound, (NO)Mn(II)TE-2-PyP4+.1

In vivo both N-methylated and N-ethylated ana-logues, Mn(III)TM(E)-2-PyP5+, offer remarkable pro-tection in different models of oxidative stress injuries[27,28,30,31,36–38], most notably in a stroke rodentmodel [37]. In biological systems Mn(III)TM(E)-2-PyP5+ may readily be reduced at the metal site [39]. Itspara analogue, Mn(II)TM-4-PyP4+, that has 160 mVmore negative E1/2, reduces O2

�− with a rate constantof 4×109 M−1 s−1 [73]. Marcus equation predicts atenfold increase in a rate constant for each 120 mVincrease in E1/2 and is shown to be valid for rate-deter-mining step of O2

�− oxidation by Mn(III) porphyrins(Fig. 4 and Ref. [27]). Assuming Marcus equation isequally valid for O2

�− reduction, the rate constant forthe O2

�− reduction by Mn(II)TM-2-PyP4+ would be�3×108 M−1 s−1. Hence, Mn(II)TM(E)-2-PyP4+

would be more reactive towards O2�− than towards

NO�; and it could function as superoxide reductase in asimilar manner suggested recently for Cu,Zn–SOD anddesulfoferredoxin [100,101]. However, given the highNO�/O2

�− ratio (�100 under normal physiologicalconditions [102], and even higher under pathologicalconditions [103,104]), along with easily reducibleMn(III) center, nitrosylation [39] may become an alter-native mode of action of Mn(II)TM(E)-2-PyP4+.Therefore, it appears obvious that tissue localization ofthe compound, concentration and the nature of thespecies the Mn complex may encounter would deter-mine its true mechanism of action.

It has been reported that Mn(III)salen+ is protectivein several models of oxidative stress [21,22]. However,Mn(III)salen+ is only modestly stable and both EDTAand Chelex are capable of dechelating it as observed byUV–Vis spectroscopy. This is consistent with the ob-served loss of SOD activity of Mn(III)salen+ in thepresence of EDTA [33], and indicates that the Mn-based antioxidants may act in vivo through a ‘free’ Mnrelease mechanism. Another piece of evidence comesfrom our E. coli studies [45]: the addition of MnCl2 tothe minimal medium [105], even at submicromolar con-centrations, allows the SOD-deficient strain of E. coli togrow under aerobic conditions [28,45]2. The cyclicvoltammetry of Mn(II) salts shows that the oxidationof manganese(II) in 0.05 M aqueous tris buffer, pH 7.8,occurs at a potential of � +450 mV versus NHE. Theoxidation of the Mn(II) is independent of its counterion, but dependent upon the hydrogen ion concentra-tion. Therefore we have attributed this oxidation waveto the Mn(II)/Mn(III)(OH) redox couple [44]. The po-

1 Total concentrations in 0.05 M phosphate buffer, pH 7.8 are 10�M GSNO, 10 �M Mn(III)TE-2-PyP5+, and 1 mM ascorbic acid.

2 At 25 �M concentrations Mn(III)TE-2-PyP5+ and MnCl2 wouldboth allow SOD-deficient E. coli to grow in casamino acids-mediumas successfully as the SOD-proficient strain.


Scheme 3. The reactions of Mn(III)TM(E)-2-PyP5+ and its one-electron reduced (Mn(II)TM(E)-2-PyP4+) and one-electron oxidized(O�Mn(IV)TM(E)-PyP4+) forms with biologically active species. (a) Data from Ref. [91]. (b) The N-methylated and N-ethylated analoguesexhibit similar SOD-like activity, have equal metal-centered redox potentials [27,28,34] and are equally sensitive to H2O2 [27,28]. Therefore, weassume they would have equal reactivities towards other reactive oxygen species, such as NO� [39] and ONOO− [32]. (c) The reported data forthe reaction of para N-methylated isomer with O2 [40] is used to estimate the reactivity of the ortho isomers, Mn(III)TM(E)-2-PyP5+. (d) The rateconstant for the oxidation of Mn(II)TM-4-PyP4+ by O2

�− is 4×109 M−1 s−1 [73]. This rate constant and Marcus equation (120 mV increasein E1/2 causes tenfold increase in a rate constant) [81] are used to calculate the rate constant for the oxidation of Mn(II)TM(E)-2-PyP4+ by O2

�−.

tential of that couple would allow, at least by thermo-dynamic criteria, a facile coupling with O2

�− reduction.Indeed, the kcat for MnCl2 in 0.05 M phosphate buffer,pH 7.8 determined by cytochrome c assay and pulseradiolysis was 1.3×106 and 1.9×106 M−1 s−1, respec-tively [33]. Further on, Lactobacillus plantarum, one ofthe few organisms lacking superoxide dismutase utilizesa high intracellular level of manganese in scavengingO2

�− [106,107]. Finally, Chiueh and coworkers [42,43]reported that manganese protects nigral neurons fromiron-induced oxidative injury and inhibits lipid peroxi-dation of brain homogenates. Therefore, a release ofmetal, which would be then active in its ‘free’ form (i.e.hydroxo species) or bound to proteins or other biologi-cal ligands, needs attention as a possible in vivo modeof action of Mn complexes.

Further work is in progress [63] on solution chem-istry of a variety of Mn(III) complexes as well as on

their application in different in vivo models of oxidativestress with emphasis on the differences in their struc-tures/reactivities and lipophilicities.

5. Abbreviations

pH*, an asterisk is used to indicate that the pH refersto an infinitely diluted solution in 9/1 methanol +aqueous solvent system rather than to the infinitelydiluted solution in water, for details see also Ref. [33];E. coli, Escherichia coli ; O2

�−, superoxide anion; NO�,nitric oxide; CO, carbon monoxide; ONOO−, peroxyni-trite; GSNO, S-nitrosoglutathione; AA, ascorbic acid;SOD, superoxide dismutase; DMF, N,N �-dimethylfor-mamide, NHE, normal hydrogen electrode;Mn(III)TPP+, manganese(III) 5,10,5,20-tetrakis-(phenyl)porphyrin; Mn(III)T(PFP)P+, manganese(III)


Fig. 4. Reactivity of manganese(III) porphyrins, manganese(III) phthalocyanine and Mn(III) tetrakis(2,3-pyridino)porphyrazine as catalysts forO2

�− dismutation, expressed in terms of log kcat(O2�−) (Table 3), plotted as a function of metal-centered redox potential, E1/2 (Table 3).

5,10,15,20-tetrakis(pentaflurophenyl)porphyrin; Mn(III)-T-2(4)-PyP+ manganese(III) tetrakis, 5,10,15,20-te-trakis(2(4)-pyridyl)porphyrin; Me, methyl; Et, ethyl;Mn(III)TM(E)-2(3,4)-PyP5+, manganese(III) 5,10,15,20 - tetrakis(N - methyl(ethyl))pyridinium - 2(3,4) - yl)-porphyrin, Mn(II)Br8TM-4-PyP4+, manganese(II)2,3,7,8,12,13,17,18 - octabromo - 5,10,15,20 - tetrakis(N-methylpyridinium-4-yl); Mn(III)Cl4TE-2-PyP5+, man-ganese(III) beta-tetrachloro34-5,10,15,20-tetrakis(N-ethylpyridinium-2-yl); Mn(III)Cl5TE-2-PyP5+, man-ganese(III) beta-pentachloro82-5,10,15,20-tetrakis(N-ethylpyridinium-2-yl); Mn(III)BM-2-PyP3+, man-ganese(III) 5,10,15,20-bis(2-pyridyl)-bis(N-methylpyri-dinium-2-yl)porphyrin; Mn(III)TrM-2-PyP4+, 5-(2-pyridyl) - 10,15,20 - tris(N - methylpyridinium - 2 - yl)-porphyrin; Mn(III)T(TMA)P5+, manganese(III)5,10,15,20-tetrakis(N, N, N-trimethylanilinium-4-yl)poprhyrin; Mn(III)T(TFTMA)P5+, manganese(III)5,10,15,20-tetrakis(2,3,5,6-tetrafluoro-N, N, N-trimethylanilinium-4-yl)poprhyrin; Mn(III)TCPP3−,manganese 5,10,15,20-tetrakis(4-carboxylatophenyl)-porphyrin; Mn(III)T(2,6-Cl2-3-SO3-P)P3−, mangane-se(III) 5,10,15,20-tetrakis(2,6-dichloro-3-sulfonato-phenyl)porphyrin; Mn(III)T(2,6-F2-3-SO3-P)P3−, man-ganese(III) 5,10,15,20-tetrakis(2,6-difluoro-3-sulfona-tophenyl)porphyrin; Mn(III)Pc+, manganese(III)phthalocyanine; Mn(III)T-2,3-PyPz+, manganese(III)tetrakis(2,3-pyridino)porphyrazine; Mn(III)T-3,4-PyPz+ manganese(III) tetrakis(3,4-pyridino)-porphyrazine; {Mn(III)BV2−}2, manganese(III) biliv-erdin IX; {Mn(III)BVDME}2, manganese(III) biliv-erdin IX dimethyl ester; {Mn(III)MBVDME}2,manganese(III) mesobiliverdin IX dimethyl ester;{Mn(III)BVDT2−}2, manganese(III) biliverdin IX di-taurate; H4BVDME+, biliverdin IX dimethyl ester.

H4BV+, 1+ charge when all propionic acid groups areundissociated (pKa of the pyrrolic nitrogens are �3[75b,33]), 5− charges when ligand is fully deproto-nated at pyrrolic nitrogens and propionates (pH 7.8).

Acknowledgements

We are grateful to Irwin Fridovich for his guidanceand support. We thank Jerry C. Bommer for the gener-ous gift of mesobiliverdin IX dimethyl ester and biliv-erdin IX ditaurate and Peter Hambright and PedatsurNeta for motivating discussions. We are also grateful toGerrado-Ferrer-Sueta for critical reading of themanuscript. The financial support from Aeolus/Incaraand Duke Comprehensive Cancer Center, grant P30CA 14236 is greatly appreciated.

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manganese(iii) complexes with porphyrins and related compounds as catalytic scavengers of superoxide

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