Superoxide-dependent redox cycling of citrate-Fe3+: Evidence for a superoxide dismutaselike activity

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 253, No. 1, February 15, pp. 257-267,1987

Superoxide-Dependent Redox Cycling of Citrate-Fe3+: Evidence for a Superoxide Dismutaselike Activity’ GIORGIO MINOTTI’

AND

STEVEN

D. AUST3

Center for the Study of Active %gen in Biology and Medtine, and Department of Biochemistry, Michigan State University, East Lmzsing, Michigan .@82&1919 Received September

15,1986, and in revised form November

14,1986

Citrate-Fe3+, reportedly a physiological chelate, exhibits superoxide dismutaselike activity, as evidenced by (i) the inhibition of xanthine oxidase-dependent cytochrome c reduction; (ii) the dismutation of xanthine oxidase-generated superoxide to hydrogen peroxide and oxygen, and (iii) the enhanced disproportionation of potassium superoxide. The catalytic activity of citrate-Fe3+ corresponds, on a molar basis, to 0.03% of that of copper- and zinc-containing superoxide dismutase. Although weak, this activity enables citrate-Fe3+ to inhibit superoxide and ADP-Fe3+-dependent peroxidation of extracted microsomal lipids. Also, the dismutase activity of citrate-Fe3+ interferes with its ability to promote lipid peroxidation. It is proposed that chelation of Fe3+ by citrate may represent a protective mechanism against the deleterious consequences of superoxide generation. 0 1987 Academic Press. Inc. The superoxide radical (0~)~ is generated by aerobic cells during several both enzymatic and nonenzymatic reactions. The biological fate of Og and its potentially deleterious impact on cell homeostasis are mediated by transition metals like copper, iron, or manganese. The reactions of these metals with 0~ are very complex and may result in two distinct, opposite processes: (i) the attenuation of 0; toxicity, or (ii) the enhancement of Og toxicity. The first process is brought about by CI.?+,Mn3+, or Fe3+ i This work was supported in part by Grant GM33443 from the National Institutes of Health. ’ Fellow of Associazione Italiana Ricerca sul Cancro and the Council for International Exchange of Scholars (Fulbright Program). Permanent address: Institute of General Pathology, Catholic University, Rome, Italy. ’ To whom correspondence should be addressed. ’ Abbreviations used: Hepes, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid; HRP, horseradish peroxidase; HzOz, hydrogen peroxide; KOz, potassium superoxide; MDA, malondialdehyde; 01, superoxide; Se-GSHPx, selenium-dependent glutathione peroxidase; SOD, superoxide dismutase.

at the active site of the various SODSwhich catalyze the dismutation of 0; to HzOz and Oa (1). As a common feature, the catalytic cycle of SOD consists of the reduction-oxidation of the metal center (Mla+‘) through subsequent encounters with 0; (2): Mn+‘+O;+Mn+Oz PI M” + 0; + 2H+ --t Mn+’ + Hz02. PI Other enzymes, namely Se-GSHPx and catalase, would in turn reduce H202 to water or water and molecular oxygen, respectively, thus completing the detoxification chain initiated by SOD (3). The second process, i.e., the enhancement of 0~ toxicity, is thought to occur via the so-called iron-catalyzed Haber-Weiss reaction. According to the proposed scheme (4), Fe3+, or analogous redox active metal in its high-valence state, is first reduced by 0; and then reoxidized by H202 Fe3++ 0; + Fe’+ + 0 2 r31 205 + 2H+ + H202 + O2

Fe2++ H202 + Fe3++ ‘OH + OH-. 257

[41

[5]

0003-9861/87 $3.00 Copyright All rights

0 1987 by Academic Press, Inc. of reproduction in any form reserved.

258

MINOTTI

The reductive cleavage of HzOzby Fe’+, also referred to as Fenton’s reaction (5), generates the ‘OH radical, which is far more reactive than 0; and may attack a multitude of biomolecules, including proteins, nucleic acids, and unsaturated lipids. In this context, the protective role of SOD consists of preventing the O;-dependent reduction of Fe3+ to Fe’+, thus precluding the latter from participating in Fenton’s reaction. The excessive generation of 0; has been implicated in the pathogenesis of metabolic, degenerative, and inflammatory diseases. Thus, it is not surprising that the pharmacological administration of SOD has been proposed as a novel therapy for these diseases (6). Interestingly, Archibald and Fridovich (7) have shown that the oxygen-tolerant, SOD-deficient Lactobacilh plantarum accumulates Mn2+ to an intracellular concentration in excess of 25 KIM. The physiological significance of this phenomenon is to provide the cell with a pool of Mn2+ complexes that scavenge 0; and allow the microorganism to survive in oxygen-saturated environments even in the absence of SOD. This finding is extremely important, since it indicates that the unavoidable dilemma of aerobic life (oxygen utilizationoxygen toxicity) may induce the cell to spontaneously evolve low-molecularweight metal complexes that mimic the activity of SOD. The characterization of these metallocomplexes would provide us with a physiological model to look at for a successful pharmacological approach to oxygen-mediated diseases. In the present work we have investigated the ability of citrate-Fe3+ to catalyze the dismutation of OZ. Citrate is a rather strong metal chelator (8); importantly, significant traces of citrate-chelated iron have been found in the low-molecularweight cytosolic fraction of rat erythrocytes and hepatocytes (g-10). Chelation of iron by citrate appears therefore to be a most likely physiological event in viva. We have found that citrate-Fe3+ catalyzes the dismutation of 0; at a rate that corresponds, on a molar basis, to 0.03% of the activity of copper- and zinc-containing SOD. Using microsomal phospholipid li-

AND

AUST

posome peroxidation as a model system we have also found that the apparently weak SOD-like activity of citrate-Fe+3 may be of importance in protecting a biological target from O;-dependent, iron-catalyzed toxic reactions. MATERIALS

AND

METHODS

Chemicals Horse heart type VI cytochrome c, 4aminoantipyrine, 2-amino-2-methyl-1-propanol, ADP, potassium superoxide, 2-thiobarbituric acid, butylated hydroxytoluene, and l,lO-phenanthroline were purchased from Sigma Chemical Co. (St. Louis, MO). Sodium citrate and phenol were products of Mallinckrodt Chemical Works, Inc. (St. Louis, MO). Sephadex G25 was obtained from Pharmacia Fine Chemicals (Piscataway, NJ). Chelex 106 ion-exchange resin (BioRad Laboratories, Richmond, CA) was used to remove trace contaminating metals from buffers and solutions. Unless otherwise specified, all the experiments were carried out in 50 rnrd NaCl carefully adjusted to pH 7.0 just prior to use. This was done because most commonly used buffers, like phosphate, Tris, and, to a minor extent, Hepes, are metal chelators (11, 12) and may therefore interfere with iron-catalyzed reactions. Tris and Hepes are also known as free radical scavengers (13, 14). Although unbuffered, the pH of the reaction mixtures did not vary from 7.0 throughout the incubation time. Enzymes. Buttermilk grade I xanthine oxidase (EC 1.1.3.22), bovine erythrocyte copper- and zinc-containing SOD (EC 1.15.1.1), bovine liver catalase (EC 1.11.1.6), and type VI salt-free horseradish peroxidase (EC 1.11.1.7) were products of Sigma Chemical Co. (St. Louis, MO). Gel-filtration chromatography on Sephadex G-25, previously equilibrated with 0.3 M NaCl, pH 7.0, was utilized to remove the antioxidant thymol and ammonium sulfate from catalase and xanthine oxidase, respectively. Following chromatography, xanthine oxidase activity was measured by aerobic reduction of cytochrome c(l), 1 unit of activity being defined as 1 rmol of cytochrome c reduced ml-’ mini; catalase was assayed by the procedure of Beers and Sizer (15). Superoxide dismutase was assayed by the method of McCord and Fridovich (1). Lyophilized commercial CuZnSOD contains traces of phosphate buffer salts. In consideration of this, some SOD preparations were incubated on ice with 10 mM EDTA for 1 h and then passed over a Sephadex G-25 column. This treatment ensured the desalting of SOD and the removal of loosely associated metals. The effects of chromatographed and unchromatographed SOD on the various assay systems were virtually identical. This indicated that contamination of SOD by phosphate buffer salts was very low and did not interfere with the reactions under investigation.

SUPEROXIDE

DISMUTATION

Ferric chelate preparation Stock solutions of ferric chelates were prepared daily by dissolving FeC& (10 mM final concentration) in 11 mM citrate or ADP. The pH’s of citrate-Fe*+ and ADP-Fe% were then carefully adjusted to 7.0 by the addition of NaOH. @tochrome c reduction Xanthine oxidase-dependent, Oi-mediated reduction of cytochrome c was monitored spectrophotometrically as the increase in absorbance at 550 nm (1). This spectrophotometric assay, like the others presented in this paper, was carried out in a Cary 219 spectrophotometer, using lcm pathlength cuvettes. Typical incubation mixtures contained 0.33 mM xanthine and 0.1 mM cytochrome c in 50 mM NaCl, pH 7.0; reactions were started by the addition of xanthine oxidase (0.050 U/ml) and the cuvette chamber was maintained at 37°C. All other additions are specified in the figure legends. Since commercially available preparations of cytochrome c are often contaminated by cyanide-inhibitable cytochrome c oxidase (16), control experiments were performed in which cytochrome e was chemically reduced by sodium dithionite, in the presence or absence of cyanide. No reoxidation of dithionite-reduced cytochrome c was observed, nor was the overall extent of reduction affected by the addition of cyanide, thus indicating that cytochrome c was essentially free of cytochrome e oxidase activity. Assay for xanthine oxiduse-dependent H&formation The formation of Hz02 in the xanthine-xanthine oxidase system was measured by a continuous spectrophotometric assay adapted, with modification, from Green and Hill (1’7). Incubation mixtures contained 0.33 InM xanthine, 5 mM phenol, 1 m&r 4-aminoantipyrine, and 4 U/ml of HRP. Reactions were initiated by the addition of xanthine oxidase (0.050 U/ml) and the cuvette chamber was maintained at 37°C. The HRP-catalyzed, HzOz-dependent oxidation of phenol and 4-aminoantipyrine gave rise to a quinone-imine adduct, the absorbance of which was monitored continuously at 505 nm. The rate of increase in absorbance at 505 nm was converted to nmol HzOz min-’ ml-’ by preparing a standard curve with known amounts of HzOz. Other additions are specified in the figure legend.

Assay for xanthine oxiduse-dependent HRP wmpound IIrformation The formation of HRP compound III in reaction mixtures containing xanthine, xanthine oxidase, and HRP was detected by difference spectroscopy as described in (18). The concentrations of HRP, xanthine, and xanthine oxidase are given in the figure legend. Spectraphotometric study of 0; d&mutation Potassium superoxide solutions were prepared by dissolving 75 mg of KOz in 25 ml of ice-cold 50 mbf NaOH, as described by Marklund (19). After 30 s, aliquots (5 ~1) of these stock solutions were immediately transferred into quartz cuvettes containing 1 ml of 50 mM 2-amino2-methyl-1-propanol, pH 9.5, and the decrease in ab-

BY CITRATE-FE3+

259

sorbanee at 250 nm was monitored continuously; the cuvette chamber was maintained at 37°C. Catalase (500 U/ml) was also added to prevent any spectrophotometric interference from HsOz. Other additions are specified in the figure legend. Iron reduction Ferric-chelate reduction was measured using the procedure of Brumby and Massey (20), with modifications (21). Aliquots (0.5 ml) of aerobic incubation mixtures of xanthine, xanthine oxidase, plus the specified ferric-chelate in 50 mrd NaCl, pH 7.0, 37”C, were sampled periodically and mixed vigorously with 1 ml of 15 mM, l,lO-phenanthroline. Trichloroacetic acid (0.2 ml of a 30% solution) was then added and the solution was mixed again. The phenanthroline complex was subsequently extracted from the aqueous phase with 2 ml of n-amyl alcohol. Following low-speed centrifugation the absorbance of the organic phase was read at 510 nm against a reagent blank. The absorbance values at 510 nm were converted to nmol Fea+ reduced ml-’ min-’ using a standard curve prepared with known concentrations of the ferric-chelate solution chemically reduced with thioglycolic acid. Preparation of microsomes, lipids, and liposomes. Male Sprague-Dawley rats (250-280 g) were obtained from Charles River (Boston, MA). Liver microsomes were isolated as per Pederson and Aust (22); lipids were extracted from freshly prepared microsomes according to Folch et al (23). All steps were performed at 4”C, with argon-purged buffer and solvents, in order to minimize the autoxidation of unsaturated lipids. Extracted lipids were stored in argon-saturated CHCls:CHsOH (2:l) at -20°C. The lipid phosphate content was assayed according to the procedure described by Bartlett (24). Liposomes were prepared by indirect anaerobic sonication of microsomal lipids as described by Pederson et al. (25). Lipid permidatim assay. Liposomes (1 cmol lipid phosphate/ml) and 0.33 mM xanthine were incubated at 37°C in 50 mM NaCl, pH 7.0, under an air atmosphere, in a Dubnoff metabolic shaker bath. Reactions were started by the addition of xanthine oxidase (0.050 U/ml). All other additions are specified in the table legend. Aliquots of the incubation mixtures were withdrawn periodically and assayed for MDA formation by the thiobarbituric acid test (26). Butylated hydroxytoluene was added to the thiobarbituric acid reagent to prevent iron-catalyzed decomposition of lipid hydroperoxides and MDA formation during the heating step of the assay (26). RESULTS

The Effect of Citrate-FeS+ Dependent Q&chrome

on Superoxidec Reduction

The time courses of xanthine oxidasedependent, Oh-mediated reduction of cytochrome c are shown in Fig. IA. Two dis-

MINOTTI

AND

AUST

0 :’ 123456

.

,

123456 TIME

TIME (mm)

(min)

FIG. 1. The effects of citrate-Fe’+ and CuZnSOD on xanthine oxidase-dependent cytochrome c reduction. (A) Incubation mixtures (1 ml, final volume) contained 0.1 mM cytochrome e, 0.33 mM xanthine, and 50 mM NaCl, pH 7.0, at 3’7’C. Reactions were initiated by the addition of xanthine oxidase (0.050 U). (---) Indicates the addition of 0.1 mM citrate-Fe’+ (0.11 mM citrate:O.l mM Fe&) or 10 U of CuZnSOD, ( * * * ) indicates the addition of 0.4 mM citrate-Fe3+ (0.44 mM citrate:0.4 mM FeCle) or 40 U of CuZnSOD. (B) All experimental conditions were as in (A) with the exception that catalase (500 U) was included.

tinct phases may easily be distinguished: (i) the rapid increase in absorbance at 550 nm, which is indicative of the reduction of ferricytochrome c, and (ii) the subsequent rapid decrease in absorbance, which is indicative of ferrocytochrome c reoxidation. When catalase was included in the reaction mixture (Fig. 1B) the reoxidation phase was eliminated, this finding is in agreement with previously published reports (21, 27) and indicates that ferrocytochrome c is oxidized by H202, which is also generated during the xanthine oxidase reaction. In the absence of catalase (Fig. 1A) the addition of increasing concentrations of citrate-Fe3+ inhibited the initial rate of cytochrome c reduction and accelerated the subsequent HzOz-dependent oxidation of ferrocytochrome c. These effects are similar to those exerted by SOD (see also Fig. 1A) and would suggest that citrate-Fe3+ may interfere with OI-dependent reduction of cytochrome c in two different ways: (i) by competing with cytochrome c for 05, and (ii) by increasing the dismutation rate of 0; to H202, thus favoring the HzOz-dependent reoxidation of ferrocytochrome c. In the presence of catalase, citrate-Fe3+ only inhibited the initial rate of OH-dependent reduction of cytochrome c, in a concentration-dependent fashion which mim-

icked that of SOD (Fig. 1B). A titration of the inhibitory effects of citrate-Fe3+ and SOD on the reduction of cytochrome c showed that, under these experimental conditions, half-maximal inhibition was achieved by either 0.1 mM citrate-Fe3+ or 10 U/ml of SOD (Fig. 2). The effects of citrate-Fe3+ and SOD were additive. In fact, as shown in Table I, the simultaneous addition of 0.1 mM citrate-Fe3+ and 10 U/ml

H

SOD (U/ml)

5

IO

20

40 M)

loo

.05

.I

.2

.4

IL0

100.

$ F m zj 50. z -e 0

.6

c--oCITRATE-Fe3+(mM)

FIG. 2. Concentration-dependent inhibition of cytochrome c reduction by citrate-Fe’+ (1.1 citrate:1 FeC& molar ratio) or CuZnSOD. Incubation mixtures were prepared as in Fig. 1B. Values are given as percentage of inhibition of the initial rate of cytoehrome c reduction.

SUPEROXIDE TABLE

DISMUTATION

Fe3+ affected the activity of xanthine oxidase, measured at 290 nm as the formation of uric acid (data not shown).

I

THE ADDITIVE EFFECTS OF CITRATE-Fee+ AND SOD ON XANTHINE OXIDASE-DEPENDENT CYTOCHROME c REDUCTIONS

Addition Xanthine oxidase Xanthine oxidase, citrate-Fe’+ Xanthine oxidase, SOD Xanthine oxidase, citrate-Fee+, SOD

The Efect of C&rate-F8 on Xanthine Oxidase-Dependent H202 Forwmtion

5% Inhibition

AAd min

Xanthine oxidase-generated HzOz was found to promote the HRP-catalyzed oxidation of phenol and 4-aminoantipyrine, measured as the increase in absorbance at 505 nm. The reaction was greatly stimulated by citrate-Fe3+ and SOD (Fig. 3). For example, 1 mM citrate-Fe3+ or 100 U/ml of SOD increased the absorbance rate at 505 nm from 0.107 to 0.223 min-‘; this corresponded to an increase in HzOzformation from 19.6 to 40.8 nmol ml-’ min-‘. Figure 3 (insert) also shows that nearly half-maximal stimulation of HzOz formation was achieved by 10 U/ml of SOD or by 0.1 mM citrate-Fe3+, i.e., the same concentrations required to inhibit by 50% the OF-dependent reduction of cytochrome c. When citrate-Fe3+ (0.1 mM) and SOD (10 U/ml) were included simultaneously in the reaction mixture, an additive stimulation of HzOz formation was observed (Table II).

0.92 0.46 0.46

50.0 50.0

0.07

92.4

a Reaction mixtures (1 ml, final volume) contained 0.1 mM cytochrome c, 0.33 mM xanthine, and catalase (500 U) in 50 mM NaCl, pH 7.0, at 37°C. Where indicated, additions were made as follows: xanthine oxidase (0.050 U); citrate-Fe’+ (0.11 mM:O.l mM); CuZnSOD (10 U). Values are given as initial rates of absorbance increase at 550 nm.

of SOD caused a 92% inhibition of the initial rate of cytochrome c reduction. Citrate alone (from 0.05 to 1 IMM) had no significant effect on the rate of xanthine oxidase-dependent cytochrome c reduction (data not shown). Also, neither citrate nor citrate-

wSOD IO

20

261

BY CITRATE-FE’+

(U/ml)

40

60

0

ICKI

.I

1.0

0 .I

.2

.4 o-CITRATE-Fd+

.6

1.0

(mM)

FIG. 3. The effect of citrate-Fe’+ (1.1 citrate:1 FeCla molar ratio) or CuZnSOD on xanthine oxidasedependent HaOa formation. Reaction mixtures (1 ml, final volume) contained 5 mM phenol, 1 mM 4aminoantipyrine, HRP (4 U), and 0.33 mM xanthine in 50 mM NaCl, pH 7.0, at 37°C. Reactions were initiated by the addition of xanthine oxidaee (0.050 U). The insert shows the concentration-dependent effect of both citrate-Fee+ and CuZnSOD on HeOa formation given as percentage of stimulation.

MINOTTI TABLE

II

THE ADDITIVE EFFECTS OF CITRATE-Fe*+ AND SOD ON XANTHINE OXIDASE-DEPENDENT H,Oa FORMATIONS

Addition Xanthine oxidase Xanthine oxidase, citrate-Fe*+ Xanthine oxidase, SOD Xanthine oxidase, citrate-Fe’+, SOD

A-&d min

76 Stimulation

0.11 0.16 0.16

45.5 45.5

0.21

90.9

DReaction mixture (1 ml, final volume) contained 5 mM phenol, 1 mM 4-aminoantipyrine, HRP (4 U), and 0.33 mM xanthine in 50 mM NaCl, pH 7.0, at 37°C. Where indicated, additions were made as follows: xanthine oxidase (0.050 U); citrate-Fe*+ (0.11 mM:O.l mM); CuZnSOD (10 U). Values are given as initial rates ofabsorbanceincreaseat505nm.

Since xanthine and urate potentially may be peroxidized by HRP (28), and therefore compete with phenol and 4-aminoantipyrine, control experiments were performed in which acetaldehyde replaced xanthine as a substrate for xanthine oxidase. The results of these experiments were in substantial agreement with those reported in Fig. 3 (data not shown). Neither phenol (5 mM) nor I-aminoantipyrine (1 mM) had any significant effect on the activity of xanthine oxidase, measured either as uric acid formation or cytochrome c reduction, respectively (data not shown).

AND

AUST

change was characterized by a sharp peak at 418 nm and a trough at 390 nm, both consistent with the spectrophotometric features of compound III (18, 33). The intermediacy of 0~ in the formation of the observed spectrum was demonstrated by the inhibitory effect of SOD or, once again, citrate-Fe3+. Since compound III is less reactive than other forms of HRP (34), control experiments were performed to verify whether the formation of this oxyperoxidase affected the HRP-catalyzed assay for HzOz. As shown in Table III, the HRP-catalyzed oxidation of phenol and 4-aminoantipyrine was initiated by the addition of H202 or xanthine oxidase. When HzOzand xanthine oxidase were included simultaneously in the reaction mixture, the increase in absorbance at 505 nm was fully additive, both in the presence and absence of citrate-Fe3+ or SOD. This indicated that, under the experimental conditions used in this study, the flux of 0; from xanthine oxidase and the consequent formation of compound III did not affect the ability of HRP to utilize exogenous Hz02 as an oxidant for phenol and 4-aminoantipyrine. The E#ect of Citrate-FeS+ cm the Dismutation of Potassium SuFoxide The dismutation of 0~ was studied by a direct spectrophotometric assay relying

The Eflect of Citrate-FeS+ on SuperoxideDependent Formation of HRP compound III Horseradish peroxidase is known to react with 0; to yield a compound III which is also referred to as oxyperoxidase (2932). Direct spectrophotometric evidence for the formation of compound III during the xanthine oxidase reaction is given in Fig. 4. Horseradish peroxidase was incubated with xanthine and xanthine oxidase and then rapidly scanned from 700 to 350 nm against a reagent blank containing only xanthine and HRP. The resulting spectral

400

500

600

700

WAVELENGTH

FIG. 4. Xanthine oxidase-dependent formation of HRP compound III. The complete reaction mixture (1 ml, final volume) contained HRP (75 U), xanthine (0.33 mM), and xanthine oxidase (0.050 U) in 50 mM NaCl, pH 7.0, at 37% (---) Indicates the addition of citrateFe*+ (1.1 mhf citrate:1 mM FeC&) whereas ( - * * ) indicates the addition of CuZnSOD (100 U). The reference cuvette contained only HRP, xanthine, and NaCl.

SUPEROXIDE TABLE

DISMUTATION

III

THE EFFECT OF HsOs AND XANTHINE OXIDASE ON HRP-CATALYZED OXIDATION OF PHENOL AND 4-iiMINOANTIPYRINE”

Addition

A Admin

None HsOs Xanthine Oxidase HsOa, xanthine oxidase Xanthine oxidase, citrate-FeS+ HrOc, xanthine oxidase, citrate-Fes+ Xanthine oxidase, SOD HsOa, xanthine oxidase, SOD

0.0 0.11 0.11 0.22 0.22 0.33 0.22 0.33

a Incubation mixtures (1 ml, final volume) contained 5 mM phenol, 1 rnhi 4-aminoantipyrine, HRP (4 U), and 0.33 mM xanthine in 50 mM NaCl, pH 7.0 at 37°C. Where indicated, additions were made as follows: HaOa, (20 nmol every minute); xanthine oxidase (0.050 U); citrate-Fe*+ (1.1 mM citrate: 1 rnM FeCls); CuZnSOD (100 U).

263

BY CITRATE-FE’+

absorbance at 250 nm was observed (see also Fig. 5). This indicated that the effects of citrate-Fe3+ and SOD on the dismutation of KOz were additive. The Xanthine Oxidase-Dependent &dox Cycling of Citrate-FeS+ The reaction of xanthine oxidase with xanthine catalyzed very low rates of citrate-Fe3+ reduction (Table IV). Interestingly, the addition of catalase did not exert any effect on the rate of iron reduction, thus indicating that H202 was not involved in the xanthine oxidase-dependent redox cycling of citrate-Fe3+. Table IV also shows, for a comparative purpose, the effects of xanthine oxidase on the redox state of ADP-Fe3+. Unlike citrate-Fe3+, ADP-Fe3+ was extensively reduced by xanthine oxidase. Furthermore, the addition of catalase increased the rate of ADP-Fe3+ reduction from 6.1 to 11.5 nmol ml-’ min-‘. This indicated that HzOz had an integral role in the xanthine oxidase-dependent redox cycling of ADP-Fe3+.

upon its intense absorptivity at 250 nm (19). Using KOz as a source of 0;) the spontaneous dismutation of 0; at pH 9.5 was determined to proceed with an apparent The Efect of Citrate-Fe?’ on Supertidehalf-life of about 30 s (Fig. 5). The addition Dependent Iron-Catalyzed Lipid of SOD (10 U/ml) decreased the half-life Peroxidation of 0~ from 30 to 10 s; the same result was The effect of citrate-Fe3+ on the initiaobtained by replacing SOD with 0.1 mM citrate-Fe3+ (Fig. 5). When citrate-Fe3+ (0.1 tion of superoxide-dependent, iron-catamM) and SOD (10 U/ml) were included si- lyzed peroxidation of polyunsaturated multaneously in the reaction mixture, the fatty acids was investigated in a reconstialmost instantaneous disappearance of any TABLE XANTHINE

Ferric chelate

TIME kec)

FIG. 5. The effect of SOD and citrate-Fe’+ on the dismutation of potassium superoxide. All experimental conditions were as described under Materials and Methods. (w) Control; (0) plus 10 U/ml CuZnSOD; (0) plus 0.1 m citrate-Fe*+ (0.11 rnM citratz0.1 mrd FeCla). (---) Indicates the addition of both citrate-Fe’+ (0.1 mre) and CuZnSOD (10 U/ml).

Citrate-Fe” Citrate-Fe” ADP-Fe’+ ADP-Fe’+,

IV

OXIDASE-DEPENDENT FERRIC CHELATES’

catalase ’ catalase

REDUCPION

OF

nmol Fe’+ ml-’ min-’ 0.5 0.5 6.1 11.5

“Incubation mixtures (3.5 ml, final volume) contained the specified ferric chelate (0.11 mM chelator: 0.1 mM FeCls), 0.33 mM xanthine, and 50 mM NaCI, pH 7.0, at 3’7°C. Reactions were initiated by the addition of xanthine oxidase (0.050 U/ml). Fd+ reduction was measured as described under Materials and Methods. Where indicated, catalase (500 U/ml) was added.

264

MINOTTI

tuted model system involving xanthinexanthine oxidase as a source of 0~ and microsomal phospholipid liposomes as a peroxidizable substrate. Table V shows that citrate-Fe3+ did not catalyze significant rates of O;-dependent lipid peroxidation; on the contrary, ADP-Fe3+ was an excellent catalyst of O;-dependent lipid peroxidation. Table V also shows that xanthine oxidase-dependent, ADP-Fe3+-catalyzed lipid peroxidation was inhibited by the addition of citrate-Fe3+. Under these circumstances the total amount of Fe2+ formed was 1.7 nmol ml-’ ml-‘; if corrected for the rate of citrate-Fe3+ reduction (0.5 nmol ml-’ min?), this value corresponds to a net formation of ADP-Fe’+ of only 1.2 nmol ml-’ min-’ versus a control rate of 6.1 nmol ml-’ min-‘. It would therefore appear that citrate-Fe3+, by scavenging 05, inhibited the reduction of ADP-Fe3+ and the initiation of lipid peroxidation. In agreement with this proposal, and the reported calculation, Table V indicates that 10 U/ml of SOD inhibited both ADP-Fe3+ reduction and ADPFe3+-catalyzed lipid peroxidation to the same extents as 0.1 I’nM citrate-Fe3+. DISCUSSION

The solubility constant of Fe3+ in aqueous solutions is very low; it has been TABLE

V

XANTHINE OXIDASE-DEPENDENT FERRIC-CHELATE REDUCTION AND LIPID PEROXIDATION~

Ferric chelate None Citrate-FeS+ ADP-Fe” ADP-Fe” citrate-Fe’+ ADP-Fe”, SOD

nmol Fe*+ ml-1 min-1’“)

nmol MDA ml-1 min-l(b)

0.5 6.1

0.0 0.1 1.2

1.7 1.2

0.3 0.3

a In (a) incubation mixtures (3.5 ml, final volume) contained 0.33 mM xanthine and the specified ferric chelate (0.11 mrd che1ator:O.l mrd FeCla) in 50 mM NaCl, pH 7.0, at 37°C; in (b) phospholipid liposomes (1 pmol lipid phosphate/ml) were included. Reactions were initiated by the addition of xanthine oxidase (0.050 U/ml). When indicated, CuZnSOD (10 U/ml) was added. MDA was assayed as described under Materials and Methods.

AND

AUST

reported that at neutral pH and in the absence of any chelator, Fe3+would probably exist as a colloidal gel (35). Chelation by sugars, nucleotides, or amino acids is therefore essential to increase the solubility of Fe3+. In this study we have shown that citrate-Fe3+, supposedly a physiological chelate, may be considered a potential catalyst of 0; dismutation. The ability of transition metal complexes to bring about the dismutation of 0~ may be tested in any one of the several assays originally developed for the measurement of the catalytic activity of the various SODS. These assays may be negative, in that they rely on the inhibition of a reaction by SOD; or positive, in that they rely on the stimulation of a reaction by SOD. Direct assays can also be used that rely upon the spectrophotometric measurement of the decay rate of chemically or radiolytically generated 0; (36). Our results demonstrate that citrate-Fe3+ displays SOD-like activity in three very different assay systems. Indeed, citrate-Fe3+ inhibited the xanthine oxidase-dependent, O;-mediated reduction of cytochrome c (negative assay), increased the rate of H202 formation in the xanthine oxidase reaction (positive assay), and catalyzed the dismutation and disappearance of potassium superoxide (direct assay). The observation that 0.1 mM citrate-Fe3+ inhibits by 50% the xanthine oxidase-dependent reduction of 0.1 mM cytochrome c implies that 0; reacts equally fast with the chelate as with cytochrome c. Therefore, the rate constant of the dismutation of 0; by citrate-Fe3+ is the same as calculated for the reduction of cytochrome c by 0; under comparable experimental conditions, i.e., 7 X lo5 M-l s-l (37). Keeping in mind that the rate constant of SOD-catalyzed 0; dismutation is 2 X 10’ M-’ s-l (38) it can be also calculated that the catalytic activity of citrate-Fe3+ corresponds, on a molar basis, to 0.03% of that of SOD. The stimulatory effect of citrate-Fe3+ and SOD on the xanthine oxidase-dependent H202 formation is worthy of further consideration. Xanthine oxidase is known to generate H202 in two different ways (39): (i) directly, via the divalent reduction of

SUPEROXIDE

DISMUTATION

molecular oxygen, or (ii) indirectly, via the univalent reduction of molecular oxygen to 05 and its subsequent spontaneous dismutation to HzOz. Superoxide dismutase, or SOD-like metallocomplexes, by catalyzing the dismutation of 05 should therefore increase the rate of xanthine oxidase-dependent H202 formation. If this reaction is monitored in a continuous spectrophotometric assay relying upon the HRP-catalyzed oxidation of suitable substrates, the O;-dependent formation of HRP compound III must also be taken into account. There is evidence to indicate that compound III is less reactive than other forms of HRP (34). As a matter of fact, Misra and Fridovich (28) could demonstrate that the oxidation of o-dianisidine, catalyzed by HRP and exogenously added H202, was inhibited by a concurrent flux of 05 from xanthine oxidase and that such inhibition was relieved by SOD. In agreement with these reports, the increase in HzOz detection shown in Fig. 3 might simply reflect the ability of citrate-Fe3+ and SOD to protect HRP from 0; and therefore to facilitate the reaction of HRP itself with the Hz02 that xanthine oxidase produces directly via divalent reduction of oxygen. We have shown (see Fig. 4) that xanthine oxidasegenerated 05 could react with HRP to yield compound III and that such reaction was strongly inhibited by citrate-Fe3+ and SOD. Nonetheless, we have also demonstrated that, in our experimental conditions, the formation of compound III did not interfere with the HRP-catalyzed assay for HzOz(see Table III). We therefore conclude that the increase in HzOz formation shown in Fig. 3 is not apparent. Rather, it is consistent with the ability of citrate-Fe3+ to catalyze, like SOD, the dismutation of 0~ to HzOz and, consequently, to increase the xanthine oxidase-dependent formation of HzOz via the univalent reduction of molecular oxygen. The assumption that citrate-Fe3+ is a mimic of the active site of SOD implies that 0; maintains this chelate within a continuous Fe3+ --) Fe’+ - Fe3+ conversion with no significant accumulation of Fe’+. Using xanthine-xanthine oxidase as a simultaneous source of both 0; and H202 we also

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observed that the addition of catalase, by scavenging HzOz, does not increase the reduction of citrate-Fe3+ (Table IV); this would be consistent with the view that citrate-iron, once reduced by 05, is rapidly reoxidized by another molecule of Og rather than by H202. Various factors may account for this reaction. For example, Hamm et al. (40) have shown that at neutral pH the formation of a citrate-Fe3+ complex is accompanied by the loss of four protons from citrate, whereas the formation of a citrate-Fe2+ complex is accompanied by the loss of only three protons. At pH values above 7.0 the additional release of a fifth proton from citrate-Fe3+ or a fourth proton from citrate-Fe2+ can be observed (40). In light of this report, it is conceivable that the Oz-dependent reduction of citrate-Fe3+ to citrate-Fe2+ causes the protonation of the citrate-iron complex and that the availability of H+ for citrateFe2’ facilitates its oxidation by 0; (see Reaction [2]). In Table IV we have also shown that the xanthine oxidase-dependent reduction of ADP-Fe3+, taken as a direct comparison, is substantially different from that of citrate-Fe3+. First, ADP-Fe3+ was reduced by xanthine oxidase at much higher rates than citrate-Fe3+. Second, the addition of catalase caused a 88% increase in the rate of ADP-Fe3+ reduction. These findings suggest that ADP-iron, as also proposed by others (41), is first reduced by 0; and then oxidized by Hz02 during the xanthine oxidase reaction. Complexes of copper with histidine, tyrosine, and lysine exhibit relatively high SOD-like activity, usually ranging from 4.9 to 7.5% of that of CuZnSOD (42). As previously specified, the SOD-like activity of citrate-Fe3+ is only 0.03% of that of CuZnSOD; this comparative observation may therefore raise a well founded skepticism as to the actual significance of citrate-Fe3+ as a physiological catalyst of 05 dismutation. However, it is important to point out that the utility of the aforementioned copper-amino acid complexes as in tivo biomimics of SOD is severely limited by the presence inside the cell of many substances which may tightly bind Cu2+(43,44). Once

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displaced from its original carrier, Cu2+ may undergo dramatic changes in its redox potential and may unexpectedly turn to catalyze the Haber-Weiss reaction rather than the dismutation of 0; (45). Furthermore, it should be realized that other iron chelates of putative physiological significance, like ADP-Fe3+, do not catalyze at all the dismutation of 0; (21,41). Rather, the presence of these “physiological” forms of iron in a biochemical system is almost invariably associated with the initiation of a potentially dangerous Haber-Weiss reaction. We would therefore suggest that the SOD-like activity of citrate-Fe3+, although weak at a first impression, can be better appreciated in the context of those multi-step pathological processes, like lipid peroxidation, that occur through an O;-driven iron catalysis. The first step in the initiation of lipid peroxidation is the Oz-dependent reduction of Fe3+to Fe2’. Not surprisingly, we have observed that citrateFe3+, in agreement with its SOD-like activity, does not reach any significant level of OT-dependent reduction and therefore cannot catalyze lipid peroxidation. Importantly, we have also found that citrateFe3+,by scavenging 05, can efficiently prevent the reduction of ADP-Fe3+ and the initiation of ADP-Fe3+-catalyzed lipid peroxidation, in a fashion that mimics the protective effect of SOD. Mammalian cells contain quite high concentrations of both mitochondrial and cytosolic SOD (46), the activity of which probably overshadows, under physiological conditions, that of trace amounts of citrateFe3+. However, there are certain disease states in which the integrity of the cell is threatened by an increase in 05 generation or by a decrease in SOD content (6). It has been suggested that, in these particular situations, the toxicity of 0~ is mediated by the release of iron from ferritin, leading to the increase in the intracellular content of redox-active chelatable iron (47). The actual concentration of this pool of iron is perhaps small and difficult to quantitate. In contrast, the availability of citrate to iron appears to be quite high. For example, the citrate content of rat liver, heart, kidney, and spleen varies from 7 up to 86 pg/

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g of fresh tissue (48). Citrate may therefore chelate much of the iron released from ferritin, thus providing the cell with an SODlike protective mechanism against OS-dependent, iron-catalyzed toxic reactions. ACKNOWLEDGMENT The authors thank Teresa L. Volimer secretarial assistance.

for expert

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