ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 289, No. 1, August 15, pp. !37-102, 1991
The Superoxide Dismutase Activities of Two HigherValent Manganese Complexes, Mn’” Desferrioxamine and Mn”’ Cyclam’ James D. Rush, Zofia Maskos, Biodynamics Institute and Departments Baton Rouge, Louisiana 70803-1800
and W. H. Koppeno12 of Chemistry
and Biochemistry,
Louisiana State University,
Received March 4, 1991, and in revised form April 30, 1991
A green manganese desferrioxamine complex is rapidly formed at room temperature upon stirring freshly precipitated manganese dioxide in a solution of the ligand. Spectral studies and low-temperature ESR indicate that this compound, which has been previously described as a manganese(III) complex, is better characterized as containing tetravalent manganese. The complex appears to form oligomers in solution. The extinction coefficient at 635 nm is 137 + 6 M-’ cm-’ (per manganese) at pH 7.8 and 88 f 4 M-’ s-l at pH 6.6 after purification by chromatography. The superoxide dismutase activity was measured and compared to that of mononuclear manganese(II1) 1,4,8,11-tetraazacyclodecane (cyclam). The catalytic rate constants for superoxide dismutase activity are 1.7 X 10’ Mm1 so’ and1 2.9 X 10’ M-l s-l for the desferrioxamine and the cyclam complexes, respectively. 0 1991
Academic
Press,
Inc.
There is an interest in small molecular weight transition metal complexes that catalyze the dismutation of superoxide. Ideally, these complexes should have the properties that make the Cu/Zn-, Mn-, and Fe-superoxide dismutases such exceptional catalysts (1). Factors that are important are (a) stability of the complex, (b) a coordination site occupied by water, and (c) a reduction potential that is amenable to reduction and oxidation by superoxide (2). Metal complexes do not benefit very much from electrostatic guidance, as the Cu/Zn protein does (3). As to stability, complete complexation is essential for copper because of the high catalytic activity of its aquo complex (4, 5). This is not an issue with manganese complexes; 1 This work was supported by a grant from the Council for Tobacco Research-U.S.A., Inc. ’ To whom correspondence and reprint requests should be addressed. 0003-9861/91
$3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
aqueous manganous ions cannot be oxidized by superoxide, and indeed these ions have no catalytic activity (2). Copper complexes have received more attention than any other metal complexes, probably because Cu/Zn superoxide dismutase catalyzes the disproportionation in a nearly diffusion-controlled fashion (3). A contributing factor might be the dissociation rate of coordinated water molecules from the metal ion. As has been pointed out, there is a relation between this rate and the catalytic rate constants of Cu/Zn-, Mn-, Fe-, and artificial Co/Zn-superoxide dismutases (6). Some low-molecular-weight manganese complexes are able to catalyze the dismutation of superoxide and protect superoxide dismutase-deficient organisms against oxidative stress (7,8). The mechanism by which manganese exerts this protective effect probably involves alternate oxidation and reduction of the metal complex. In this regard, superoxide anions have been shown to oxidize simple manganous complexes (9-11). A subsequent reduction by superoxide would restore the lower oxidation state. Recently, we examined the ability of a number of Mn” aminopolycarboxylate complexes to catalyze superoxide dismutation. Mn” nitrilotriacetate has a high catalytic rate constant of 2.2 X lo7 M-l s-l at pH 6, 22”C, and 10 mM ionic strength (2) which is only surpassed by a water soluble manganese porphyrin (12). The dismutation of superoxide catalyzed by (trispicolinato)manganese(I1) in dimethyl sulfoxide is an order of magnitude slower (13). A disadvantage of ethylenediamine-N,N’-diacetate and nitrilotriacetate is the need for an excess of ligand to assure substantial chelation. Fridovich and co-workers described the protective effect (14) of a superoxide dismutase mimic which is a complex of higher-valent manganese with desferrioxamine B (15). The ligand-metal interaction is strong enough not to require excess desferrioxamine, and the manganese is believed to be in the 3+ oxidation state (16, 17), analogous 97
98
RUSH,
MASKOS,
to iron(II1) desferrioxamine. Desferrioxamine by itself is known to react with superoxide (18-20). The rate of reaction was a matter of dispute (21,22), which has recently been resolved in a pulse radiolysis study. The bimolecular rate constant appears to be smaller than 2 X lo4 M-l s-l (23).
We have significantly modified the synthesis of the manganese desferrioxamine complex and introduced a chromatography step to remove minor contaminants by column chromatography. The complexed manganese is tetravalent and probably oligomeric. We have prepared manganese desferrioxamine in a solid state and measured its ability to dismutate superoxide in solution. EXPERIMENTAL
AND
KOPPENOL
at 130 K to detect signals arising from manganese(IV). Cyclic voltammetry experiments on deaerated solutions of the purified complex in 0.1 M NH&O3 were carried out at the platinum working electrode of an IBM EC/225 voltammetric analyzer. The superoxide dismutase activities of Mnn desferrioxamine and Mn”’ cyclam were measured by competition with the reduction of ferricytochrome c by superoxide (2). The concentration of ferricytochrome c was 30 HIM as determined from At(red-ox) at 550 nm of 21,100 (27). Superoxide radicals were generated by photolysis at 254 nm in quartz cuvettes of a solution of paraquat dichloride (0.50 mM) and sodium formate (0.10 M) at pH 7.2. Photolysis in aerated solutions (28) proceeds by the reactions
,Q2’ 2 .,Q2’ *PQ”
PROCEDURES
Deferoxamine mesylate (desferrioxamine), bovine superoxide dismutase, and cytochrome c (Type VI) were purchased from Sigma Chemical Co. Other reagent grade chemicals were from Fisher Chemical Co. and J. T. Baker. Brown hydrated manganese(W) oxide was freshly precipitated by the addition of ethanol to a solution of potassium permanganate cooled in ice. The precipitate was filtered, washed with water, and air dried. Hydrated manganese(IV) oxide (420 to 450 mg) was then added to 5 ml of a 0.2 M desferrioxamine solution. The reaction resulted in a green complex, and was complete within 30 min. A 10-g sample of manganese(IV) oxide that was kept for a week exposed to air lost approximately 3 g of water, and took two and a half times as long to form the green complex. For purification 2-4 ml of the green manganese desferrioxamine solution was applied to a 40 X 2.5 cm DEAE-Sephacel (anion) column and eluted with a Na,K-phosphate buffer or an ammonium bicarbonate buffer. A gradient (15 + 100 mM ionic strength) gave best results. Manganese desferrioxamine is also formed when desferrioxamine is added to a stirred manganese(H) solution buffered near neutral pH in the presence of oxygen, as evidenced by the green color and an absorption maximum at 635 nm. Manganese was determined by oxidation to permanganate with potassium periodate (24). The solid form was prepared by removing water at 0.01 atm (1 kPa) above phosphorus pentoxide. The material dried as an amorphous powder, but did not form crystals. It was macerated under acetonitrile until it formed hard black grains which were stored over phosphorus pentoxide. Several grindings under acetonitrile were necessary to remove water and decomposition products. The material was soluble in water and ethanol, but precipitated as an oil when acetonitrile was added. Reactions of benzohydroxamic acid with manganese(II1) acetate were carried out in water and in acetonitrile. In water, NaOH was used to deprotonate the benzohydroxamic acid, and at pH 8.5 it dissolves the manganic acetate to form a red-purple complex which is stable above pH 5. An organic base, piperidine, was used to effect the same reaction in acetonitrile. Evaporation of the latter yielded a dark green oil, but no crystals. Manganese(IV) oxalate was prepared and purified by a literature method (25). Its composition is given as K2Mn(C,0,)z(0H)s.Hz0. A solution in dilute oxalic acid showed no EPR signal at 120 K, which indicates that the manganese species is likely to be dimeric, and that the solid is more correctly formulated as K4Mn’V,(C,04)4(0)2. 3H20. Manganese(II1) cyclam3 was prepared by a literature method (26). Electron spin resonance spectra were recorded on a Varian ER200 spectrometer at 9.48 GHz (X-band) frequency. The samples were kept
3 Abbreviations used: DF, desferrioxamine; cyclam, 1,4,8,11-tetraazacyclodecane; PQ”, paraquat (l,l’-dimethyl-4,4’bipyridylium); cz’, ferrocytochrome c formed in the absence of a catalyst; c,” ferrocytochrome c formed in the presence of a catalyst.
+ HCO, + PQ’
[II + H+ + Co’,-
PI
Co;- + PQ’+ + CO2 + PQ’
131
PQ+ + Oz + PQ2+ + CI-
[41
Direct scavenging of COze and PQ’ by ferricytochrome c (29, 30) in an air-saturated solution amounts to about 10% of the total yield of ferrocytochrome c (-3 rM/min) as determined from an experiment in which superoxide was removed by approximately 5 pM Cu/Zn superoxide dismutase.
RESULTS
The absorption spectrum of the green solution that results from the reaction of desferrioxamine with freshly precipitated manganese(IV) hydroxide is identical to that of a solution prepared with slower dissolving, crystalline, MnOp (16), and very similar to the absorption spectrum of M#oxalate (Fig. 1). Benzohydroxamic acid complexes ferric ions to give an absorption spectrum identical in absorption maximum and extinction coefficient to ferrioxamine as shown in Fig. 2. Chromatography indicates that the compound is not pure. With a gradient of 15 to 100 mM ammonium bicarbonate three bands elute: a fast moving yellow fraction, a slower moving green fraction, followed by a deep green
1800,\
c
I I
-i 12ool \ I
FIG. 1. Absorption spectra of manganese desferrioxamine (right ordinate) compared with that of manganese(IV) oxalate in 0.01 M oxalic acid (left ordinate). The oxalate complex is probably an oxobridged dimer in solution.
SUPEROXIDE
DISMUTASE
ACTIVITY
FIG. 2. Absorption spectra
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 289, No. 1, August 15, pp. !37-102, 1991
The Superoxide Dismutase Activities of Two HigherValent Manganese Complexes, Mn’” Desferrioxamine and Mn”’ Cyclam’ James D. Rush, Zofia Maskos, Biodynamics Institute and Departments Baton Rouge, Louisiana 70803-1800
and W. H. Koppeno12 of Chemistry
and Biochemistry,
Louisiana State University,
Received March 4, 1991, and in revised form April 30, 1991
A green manganese desferrioxamine complex is rapidly formed at room temperature upon stirring freshly precipitated manganese dioxide in a solution of the ligand. Spectral studies and low-temperature ESR indicate that this compound, which has been previously described as a manganese(III) complex, is better characterized as containing tetravalent manganese. The complex appears to form oligomers in solution. The extinction coefficient at 635 nm is 137 + 6 M-’ cm-’ (per manganese) at pH 7.8 and 88 f 4 M-’ s-l at pH 6.6 after purification by chromatography. The superoxide dismutase activity was measured and compared to that of mononuclear manganese(II1) 1,4,8,11-tetraazacyclodecane (cyclam). The catalytic rate constants for superoxide dismutase activity are 1.7 X 10’ Mm1 so’ and1 2.9 X 10’ M-l s-l for the desferrioxamine and the cyclam complexes, respectively. 0 1991
Academic
Press,
Inc.
There is an interest in small molecular weight transition metal complexes that catalyze the dismutation of superoxide. Ideally, these complexes should have the properties that make the Cu/Zn-, Mn-, and Fe-superoxide dismutases such exceptional catalysts (1). Factors that are important are (a) stability of the complex, (b) a coordination site occupied by water, and (c) a reduction potential that is amenable to reduction and oxidation by superoxide (2). Metal complexes do not benefit very much from electrostatic guidance, as the Cu/Zn protein does (3). As to stability, complete complexation is essential for copper because of the high catalytic activity of its aquo complex (4, 5). This is not an issue with manganese complexes; 1 This work was supported by a grant from the Council for Tobacco Research-U.S.A., Inc. ’ To whom correspondence and reprint requests should be addressed. 0003-9861/91
$3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
aqueous manganous ions cannot be oxidized by superoxide, and indeed these ions have no catalytic activity (2). Copper complexes have received more attention than any other metal complexes, probably because Cu/Zn superoxide dismutase catalyzes the disproportionation in a nearly diffusion-controlled fashion (3). A contributing factor might be the dissociation rate of coordinated water molecules from the metal ion. As has been pointed out, there is a relation between this rate and the catalytic rate constants of Cu/Zn-, Mn-, Fe-, and artificial Co/Zn-superoxide dismutases (6). Some low-molecular-weight manganese complexes are able to catalyze the dismutation of superoxide and protect superoxide dismutase-deficient organisms against oxidative stress (7,8). The mechanism by which manganese exerts this protective effect probably involves alternate oxidation and reduction of the metal complex. In this regard, superoxide anions have been shown to oxidize simple manganous complexes (9-11). A subsequent reduction by superoxide would restore the lower oxidation state. Recently, we examined the ability of a number of Mn” aminopolycarboxylate complexes to catalyze superoxide dismutation. Mn” nitrilotriacetate has a high catalytic rate constant of 2.2 X lo7 M-l s-l at pH 6, 22”C, and 10 mM ionic strength (2) which is only surpassed by a water soluble manganese porphyrin (12). The dismutation of superoxide catalyzed by (trispicolinato)manganese(I1) in dimethyl sulfoxide is an order of magnitude slower (13). A disadvantage of ethylenediamine-N,N’-diacetate and nitrilotriacetate is the need for an excess of ligand to assure substantial chelation. Fridovich and co-workers described the protective effect (14) of a superoxide dismutase mimic which is a complex of higher-valent manganese with desferrioxamine B (15). The ligand-metal interaction is strong enough not to require excess desferrioxamine, and the manganese is believed to be in the 3+ oxidation state (16, 17), analogous 97
98
RUSH,
MASKOS,
to iron(II1) desferrioxamine. Desferrioxamine by itself is known to react with superoxide (18-20). The rate of reaction was a matter of dispute (21,22), which has recently been resolved in a pulse radiolysis study. The bimolecular rate constant appears to be smaller than 2 X lo4 M-l s-l (23).
We have significantly modified the synthesis of the manganese desferrioxamine complex and introduced a chromatography step to remove minor contaminants by column chromatography. The complexed manganese is tetravalent and probably oligomeric. We have prepared manganese desferrioxamine in a solid state and measured its ability to dismutate superoxide in solution. EXPERIMENTAL
AND
KOPPENOL
at 130 K to detect signals arising from manganese(IV). Cyclic voltammetry experiments on deaerated solutions of the purified complex in 0.1 M NH&O3 were carried out at the platinum working electrode of an IBM EC/225 voltammetric analyzer. The superoxide dismutase activities of Mnn desferrioxamine and Mn”’ cyclam were measured by competition with the reduction of ferricytochrome c by superoxide (2). The concentration of ferricytochrome c was 30 HIM as determined from At(red-ox) at 550 nm of 21,100 (27). Superoxide radicals were generated by photolysis at 254 nm in quartz cuvettes of a solution of paraquat dichloride (0.50 mM) and sodium formate (0.10 M) at pH 7.2. Photolysis in aerated solutions (28) proceeds by the reactions
,Q2’ 2 .,Q2’ *PQ”
PROCEDURES
Deferoxamine mesylate (desferrioxamine), bovine superoxide dismutase, and cytochrome c (Type VI) were purchased from Sigma Chemical Co. Other reagent grade chemicals were from Fisher Chemical Co. and J. T. Baker. Brown hydrated manganese(W) oxide was freshly precipitated by the addition of ethanol to a solution of potassium permanganate cooled in ice. The precipitate was filtered, washed with water, and air dried. Hydrated manganese(IV) oxide (420 to 450 mg) was then added to 5 ml of a 0.2 M desferrioxamine solution. The reaction resulted in a green complex, and was complete within 30 min. A 10-g sample of manganese(IV) oxide that was kept for a week exposed to air lost approximately 3 g of water, and took two and a half times as long to form the green complex. For purification 2-4 ml of the green manganese desferrioxamine solution was applied to a 40 X 2.5 cm DEAE-Sephacel (anion) column and eluted with a Na,K-phosphate buffer or an ammonium bicarbonate buffer. A gradient (15 + 100 mM ionic strength) gave best results. Manganese desferrioxamine is also formed when desferrioxamine is added to a stirred manganese(H) solution buffered near neutral pH in the presence of oxygen, as evidenced by the green color and an absorption maximum at 635 nm. Manganese was determined by oxidation to permanganate with potassium periodate (24). The solid form was prepared by removing water at 0.01 atm (1 kPa) above phosphorus pentoxide. The material dried as an amorphous powder, but did not form crystals. It was macerated under acetonitrile until it formed hard black grains which were stored over phosphorus pentoxide. Several grindings under acetonitrile were necessary to remove water and decomposition products. The material was soluble in water and ethanol, but precipitated as an oil when acetonitrile was added. Reactions of benzohydroxamic acid with manganese(II1) acetate were carried out in water and in acetonitrile. In water, NaOH was used to deprotonate the benzohydroxamic acid, and at pH 8.5 it dissolves the manganic acetate to form a red-purple complex which is stable above pH 5. An organic base, piperidine, was used to effect the same reaction in acetonitrile. Evaporation of the latter yielded a dark green oil, but no crystals. Manganese(IV) oxalate was prepared and purified by a literature method (25). Its composition is given as K2Mn(C,0,)z(0H)s.Hz0. A solution in dilute oxalic acid showed no EPR signal at 120 K, which indicates that the manganese species is likely to be dimeric, and that the solid is more correctly formulated as K4Mn’V,(C,04)4(0)2. 3H20. Manganese(II1) cyclam3 was prepared by a literature method (26). Electron spin resonance spectra were recorded on a Varian ER200 spectrometer at 9.48 GHz (X-band) frequency. The samples were kept
3 Abbreviations used: DF, desferrioxamine; cyclam, 1,4,8,11-tetraazacyclodecane; PQ”, paraquat (l,l’-dimethyl-4,4’bipyridylium); cz’, ferrocytochrome c formed in the absence of a catalyst; c,” ferrocytochrome c formed in the presence of a catalyst.
+ HCO, + PQ’
[II + H+ + Co’,-
PI
Co;- + PQ’+ + CO2 + PQ’
131
PQ+ + Oz + PQ2+ + CI-
[41
Direct scavenging of COze and PQ’ by ferricytochrome c (29, 30) in an air-saturated solution amounts to about 10% of the total yield of ferrocytochrome c (-3 rM/min) as determined from an experiment in which superoxide was removed by approximately 5 pM Cu/Zn superoxide dismutase.
RESULTS
The absorption spectrum of the green solution that results from the reaction of desferrioxamine with freshly precipitated manganese(IV) hydroxide is identical to that of a solution prepared with slower dissolving, crystalline, MnOp (16), and very similar to the absorption spectrum of M#oxalate (Fig. 1). Benzohydroxamic acid complexes ferric ions to give an absorption spectrum identical in absorption maximum and extinction coefficient to ferrioxamine as shown in Fig. 2. Chromatography indicates that the compound is not pure. With a gradient of 15 to 100 mM ammonium bicarbonate three bands elute: a fast moving yellow fraction, a slower moving green fraction, followed by a deep green
1800,\
c
I I
-i 12ool \ I
FIG. 1. Absorption spectra of manganese desferrioxamine (right ordinate) compared with that of manganese(IV) oxalate in 0.01 M oxalic acid (left ordinate). The oxalate complex is probably an oxobridged dimer in solution.
SUPEROXIDE
DISMUTASE
ACTIVITY
FIG. 2. Absorption spectra
