Document 825op6e14G3beemgx2mR1ENVy

.b. n. r R.u.dicu. l Biulug.y d.. Md.ic'iiic, Vol. 9. pp. 143-1411. 1990 0891-5849/90 $3.00+ .00 ' Peraarnon Press DIC zesis Paper MULTIPLE ACTIONS OF SUPEROXIDE DISMUTASE: WHY CAN IT BOTH INHIBIT AND STIMULATE REDUCTION OF OXYGEN BY HYDROQUINONES? BRIAN BANDY, JIM MOON, and ALLANJ. DAVISON* Biocnergrlics Research Laboralory. Faculty of Applied Sciences. School of Kinesiology, Simon Frascr University, Burnaby. B.C. V5A IS6 (Received 5 April 1990;Accepred 25 April 1990) Abstract-Superoxide dismutase can either inhibit or stimulate autoxidation of different hydroquinones. suggesting niulfiple roles for 0,.-. Inhibitory actions of superoxide dismutase include termination of 0,---propagated reaction chains and metal chelation by the apoprotein. Together, chelation of metals and termination of 0,---propagated chains can effectively prevent reduction of oxygen. Chain termination by superoxide dismutase can thus account for negligible accumulation of H,O, without invoking a super0xide:semiquinone oxidoreductase activity for this enzyme. One srimulorory action of superoxide dismutase is to decrease thermodynamic limitations to reduction of oxygen. Whether superoxide dismutase inhibits or acceleratesan autoxidation depends on the reduction potentials of the quinone and the availability of metal coordination for inner sphere electron transfers. Keywords-Hydroquinone autoxidation, Superoxide dismutase, Oxygen reduction, Metal coordination, Inner sphere electron transfers. Chain propagation, Semiquinone, Free radicals J I1 : 11 INTRODUCTION Cadenas and coworkers have recently compiled a wealth of data on multiple effects of superoxide disniutase in hydroquinone autoxidations.1,2These data complement data of Winterbourn on autoxidation of pyrimidines.'The diverse, paradoxical, and sometimes contradictory actions of superoxide dismutase have prompted an equally wide range of explanations, including the novel suggestion that superoxide dismutase has superoxide:semiquinone oxidoreductase activity.\ This suggestion rests on the observation that superoxide disniutase inhibits autoxidarion of the hydroquinone derivatives of 2-hydroxy-p-benzoquinoneand 1,4-naphthoquinones but prevents accumulation of H102th,e product of superoxide dismutation. Arguing against this suggestion however, superoxide dismutase only inhibits autoxidation of 1,4-naphthohydroquinone in the early phase.' Superoxide dismutase srimirlates the autoxidation when comproportionation of hydroquinone with quinone product becomes available to propagate the autoxidation .4Superoxide dismutase also 'Author IO whom correspondence should be addressed. does not inhibit, but accelerates oxidation of 1.2-naphthohydroquinoneand5-hydroxy-1,4-naphthohydroquinones.'s2 This wealth of recent data brings a need for some generalizations as to mechanisms by which superoxide dismutase can slow or accelerate autoxidations. Inhibitory actions of superoxide dismutase include termination of free radical chains and metal chelation by the apoprotein. Superoxide dismutase can also accelernre oxidations by at least three different mechanisms. We outline here some of the situations where superoxide dismutase might accelerate or slow hydroquinone oxidation. and offer possible explanations for some of the contrasting effects on different naphthohydroquinones. METAL CHEL4TION CAN ACT SYNERGISTICALLY WITH SUPEROXIDE DISMUTASE ACTIVITY TO INIlIBlT AUTOXIDAL'IONS Most meta1;chelating agents sensitize autoxidation of hydroquinone compounds to further inhibition by superoxide dismutase.s.6 Metal chelators often inhibit autoxidations by decreasing the ability of the metal to I44 B. BANDYP I al. gain or lose electrons. With a strong reductant such as 6-hydroxydopamine however, EDTA stimulates the tracc metal catalyzcd autoxidationfi p r c h n a b l y by accclerating the (ratc litiiiting) autoxidation of rcduced iron. Desferrioxamine, a stronger chelator of ferric ions (thus decreasing the reduction potential further), slows autoxidation of 6-hydroxydopa!nine. Either chelator sensitizes 6-hydroxydopamine autoxidation to inhibition by superoxide disrnutase.6 By preventing formation of a ternary reductant-metal-oxygen complex, these chelators prevent sequential "inner sphere" transfer of 2 electrons and force release of 02*an-d Q--.5.6TheO,-- and Q.- released then serve as reaction propagators: and Since EDTA accelerates autoxidation of ferrous ions (and thus 02.r-elease) but slows reduction of ferric ions (and thus Q.- formation), 02-is-the major prop- agator which accelerates the overall autoxidation i n the presence of EDTA. With I .2.4-l~ctizcnctriol. both t D T A itnd tlcslcrrioxniriirie stitiiulatcd thc autoxidntioti (altlicriigli desferrioxaiiiine caused a slight lag) (Fig. I ) . Despite stimulating autoxidation of I .2.4-benzenetriol when present alone, EDTA or desferrioxaiiiine sensitized the autoxidation to inhibition by small, catalytic quantities of superoxide dismutase. Togethcr, tiietal chelation and superoxide dismutase activity can prevent consumption of oxygen (Fig. 2). Metal binding by proteins, including superoxide dismutase apoprotein, can also play this sensitizing role"' (Figs. 2 and 3). Such inhibition favors the hydroquinone form and also prevents accuniulation of products, including H202,as found by Cadenas et al.' The concentrations of superoxide dismutase and total protein in thc experiments of Cadenas et al.' on autoxidation of 1,2,4,-benzenetriol (present at 20 p M ) were 3 p g / m L (9 U/riiL) and 6.5 IrgltnL (3.5 pglrnL NAD(P)H:quinone oxidoreductase stabilized with albumin). Trace metals were thus coiiipetitively bound by proteins i n these experiments, and the autoxidation was sensitive to inhibition by superoxide dismutase. A superoxide:semiquinone oxidoreductase activity could prevent the autoxidation, but so could 0.25 Absorbance (490rm) 0.2 0.15 Control So0 0.1 0.05 1EDTA + So0 0 0 40 80 120 160 200 Time (secwds) Fig. I. I .2,4-Benzenetriol autoxidation: Interactions between superoxide dismutase and metal chelators. Reactions were conducted in air-saturated, 50 mM phosphate buffer; pH 7.5; 25C. Where indicated. EDTA or desferrioxaiiiine (DEI.) were present a l 0.5 mM; superoxide dismutase (SOD)at 0.5 pglrnL (2 UlmL). Reactions were initiated hy addition of an aliquot from an anaerobically prepared stock solution of 1,2.4-benzenelriol lo give an initial concentration of 2.50 pM. Formation of the p- quinone product of oxidation was followed spectrophotometrically at 490 nm. Superoxide disnwlare i n hydroquinone autoxidations T260 240 220 I45 + so0 Oxygen (4) 200 +180 160 140 I120 0 II I I I 8I I II I I I 30 60 90 120 150 180 210 240 Time (seconds) Fig. 7. I,l,-l-Uelizenetriol autoxidation: Oxygen consumption in the presence of superoxide dismurase and metal chelators. Keactioo conditions were as in Figure I.Where indicated, albuniin was present at 26 &mL. Other reagents were at the same cullcentr;ltionr as 111 I;igure I . Oxygen consumplion was nionitored polarogrnphically upon ;iddilion of I.2.3-benzenetriol. t0.3 I 0.25 0.2 Absorbance (490~) 0.15 0.1 0.05 0 / albunin / dSW 2.5ug/ml dSo0 + So0 albunin + So0 I46 D. BANDYet al. dismutation of 02.b-y,preventing propagation of the radical chain. - SUPEROXIDE DISMUTASE CAN INHIUIT AUTUXIDATlONS A N D I-1& FOBMATlON UY CHAIN TERMlNATlqN Inhibitory actions of superoxide dismutase can result from accelerated termination of free radical chains. Most if not all autoxidations are chain reactions. The main chain propagating action of superoxide in hydroquinone oxidation is: K i = 2.1 nM)'.' suggest more effective competition for binding of metals by the adjacent phenolic hydroxyls of 2-hydroxy- I ,4-naptliohydroquinone. Thus, as with autoxidation or I .2,4-benzcnctriol. sufficient protein to compete for trace metals can liniit iiincrsphere electron transfers and sensitize the autoxidation to inhibition by superoxide dismutase. The 5-hydroxyI,4-naphthoiiydroquinone derivatives form a six-iiiembered ring with cations (thus showing strong intramolecular hydrogen bonding).'" These derivatives would bind metal cations avidly, and thus react primarily by concerted inner sphere transfer of electrons to oxygen. Inhibition by superoxide dismutase in autoxidations is usually considered evidence that superoxide is an intermediate in the free radical chain.'.R To the extent that oxidation of adrenaline is typical, superoxide has an amplification factor of up to In other words, one molecule of superoxide can stimulate oxidation of 10 molecules of adrenaline before its participation is ended through some chain termination reaction. On this basis, superoxide dismutase prevents the formation of I O molecules for every half-molecule of H202it manufactures. Thus, one explanation for the phenomena described'.' is that superoxide dismutase diminishes formation of H20,by facilitating chain termination. The combined effects of metal binding and chain termination can prevent oxygen consumption9 and thus decrease H 2 0 2production IO negligible levels. METAL CHELATION BY PROXIMAL HYDROXYL GROUPS ALLOWS INNER SPHERE AUTOXIDATION Why does superoxide dismutase not inhibit 1,2naphthohydroquinone' or 5-hydroxy-1,4-naphthohydroquinone' oxidation? That the semiquinone is not a substrate for superoxide:semiquinone oxidoreductase was offered as one explanation for the lack of inhibition of the 1,2-naphthohydroquinone autoxidation by superoxide dismutase.' An alternative explanation is the difference in metal-binding capabilities of the different hydroquinones. The o-quinol with its adjacent phenolic hydroxyls binds metals more avidly than the (para) 1,4-napthohydroquinone. Thus the o-quinol can autoxidize by an inner sphere niechanism and is less influenced by relatively weak chelators such as proteins. The much higher concentrations of superoxide dismutase needed to inhibit autoxidation of the 2-hydroxy1,4-napthohydroquinone (12 pglmL, K i = 540.8 nM)'., than the 1,4-napthohydroquinone (0.3 p g l m L , SUPEROXIDE DISMUTASE CAN ACCELERATE AUTOXI1)ATlONS BY SEVERAL MECllANlShlS Why might superoxide dismutase stiriidarP autoxidation of I ,2-naphtIiohydroquinone' and 5-hydi~oxy1,4-naphthohydroquinone~?~Stimulatory effects of superoxide disniutase on autoxidations are sonietimes reported."2,''.'L.'' 1n general, they occur in reactions: a) in which superoxide is a net terminator of free radical chains b) in which hydrogen peroxide is a better oxidant than oxygen eg. QH, + H20z- Q.- + .OH + OH- + 2ft+ Q * - + H202- Q + .OH + O H - . or c) in which semiquinone or quinone formation i: limited by accumulation of superoxide or These stimulatory effects require that they are not ovei powered by metal chelation and chain termination h superoxide dismutase. Stimulatory effects of s u p e oxide dismutase are most apparent when 02-.-depel dent propagation beconies less significant, such : when accumulation of quinone allows comproportioi ation with the h y d r ~ q u i n o n eB. ~y inhibiting chain pro agation by 02.-and stimulating semiquinoi a u t ~ x i d a t i o n , ~o. n' ~e biologically important effect superoxide dismutase is to limit formation semiquinones. Superoxide dismutase in hydroquinone autoxidalions 147 SUPEROXIDE DISMU'rASE CAN ACCELERATE 0SIL)A'I'IONS IN WHICH EQUlLlURIA FORMlNG SEhllQUlNONES OR QUINONES A R E LIMI'TED BY 'I'IIEHblODYNAMIC CONSIDERATIONS Superoxide disniutase can accelerate an autoxida- + +tion by displacing the equilibria: Q.- O2f, Q +0 2 . -in the direction of q u i n o ~ i e ? ~o' ~r Q* ~H', O2++ +Q . - O,.- in the direction of semiquinone." This effect is not seen for strong reductants with reduction potentials low enough to minimize product inhibition by OL.-(such as 6-hydroxydopamine, I ,2,4-benzenetriol or 2-hydroxy- 1,4-naphthohydroquinone). I ,2Naplithohydroiluinone is a weaker reductant than I ,4naplirholiydroquinone or 2-hydroxy-I ,4-naphthohy- +droquirione (E"'(Q/QH,) = 143 niV vs. + 3 6 mV and - 139 niV)'' and thus is more unfavorable foroneelectron reduction of oxygen, increasing product inhibition and favoring acceleration by superoxide disinutase. Similarly, 5-hydroxy-1,4-naphthohydroquinone is a weaker reductant than I ,4-naphthohydroquinone (E,,? = - 140 tiiV vs. - 180 niV)I6 and additionally binds metals strongly to favor inner-sphere electron transfers. 1,4-Benzohydroquinorie would not bind metals as strongly as I ,2,4-benzenetriol, but is a +tnucli weaker reductant (E"'(Q/QH,) = 280 mV ver+sus 106 mV)," and autoxidation was stimulated by superoxide dismutase." The contrast between 1,2-naphthoquinone and 2- hydroxy- I ,4-naphthoquinone resembles the contrast between the stimulation by superoxide dismutase of autoxidation of gallic acidi2and inhibition of autoxidation of pyrogallol. l7 These compounds have identical o-hydroxy substitutes capable of binding metal ions. tlowever, an electron withdrawing p-carboxyl substituent on gallic acid raises the reduction potential compared to pyrogallol, making it a weaker reductant. This effect slows autoxidatioii, and increases product inhibit ion. Thus on the one hand, superoxide dismutase can stitiiulate 1,2-naphthohydroquinone autoxidation by decreasing product inhibition (i.e., the 2-hydroxy-l,4napthohydroquinone is less affected by product inhibition because of the additional electron-donating hydroxy group). On the other hand, superoxide dismutase can inhibit autoxidation of 1,4-napthohydroquinone and ?-hydroxy- I ,4-napthohydroquinone by metal chelation and chain terniination. H EDUC'lION 1'01 EN'rIA LS FAV 0R OXlDATION (HA'I'HER THAN KEDUCTlON) O F S E M l Q U l N O N E UY SU1'EHOXII)E Finally, the proposed novel enzyme activity might be questioned on thermodynamic grounds. Semiqui- none:superoxide oxidoreductase activity may be more feasible than the proposed superoxide:semiquinone oxidoreducfase activity. This is because enzymes have no effect on the overall free energies of the reactions they catalyze and the most stable products are the quinone and H 2 0 z . On the Principle of Microscopic Re- '*versibiliry. t~ the extent that superoxide tlismutase accelerates the transfer of an electroll froui OL.-to Q.-, it will also facilitate transfer of an electron from Q.- to Ole-. I n a reaction mixture in which both 02.and Q.- are present, the direction of the reaction, and thus the product distribution, depend on thermodynamic considerations. Such considerations more strongly favor the formation of Q and Hz02than the reverse reaction. Although reduction of the semiquinone by superoxide may be thermodynamically feasible, other things being equal, oxidation of the semiquinone by superoxide is more favorable." Unless the enzyme were to act by a mechanism which favored one half-reaction over another (e.g., by specifically providing protons to one product), the thermodynaniics favor formation of quinone and H202. CONCLUSlON The diversity of actions of superoxide disrnutase reflects the diversity of roles played by 02.-S.uperoxide propagates or terminates free radical chains, accelerates or retards oxidation of hydroquinones o r semiquinones. The availability and coordination state of metals and the reduction potentials of the quinone and semiquinone all influence the roles which 02.plays. Such diversity offers a range of explanations for paradoxical effects of superoxide dismutase which deserve exploration. In general, we conclude: I . To the extent that reduction of oxygen occurs by sequential inner sphere electron transfers, superoxide dismutase will be without effect. 2. To the extent that reduction of oxygen by semiquinone is thermodynamically unfavorable, superoxide dismutase will accelerate hydroquinone autoxidation. 3. To the extent that reduction of oxygen by semiquinone is thermodynamically favorable, and 'An example is I .4-naphthohydroquinone, the weakest reductant tested inhibitable by superoxide dismutare (therefore most thermodynamically favored for a semiquinone reductase activity). The tworlectruii reduction potential at pH 7 is + 3 6 mV.''~'Y'rhrone-electron reduction potentials for 1 ,J-iiaphthoquinone are also known (E"' (Q.-/Q'-) = +212 mV and E"'(Q/Q.-) = - 140 mV).' Thus, rrducrioti of the semiquinone by superoxide (E"'(O,/O,.-) = - 155 mV at unit conccntration) has a deltaE"' of +367 mV. However, +o.riduiron of the semiquinone by superoxide (E"'(O,.-/H,O,) = + 865 mV) has a deltaE"' of 1005 mV. I48 E. BANDYrr al. 0 2 . - iS the major propagating species, superoxide dismutase will inhibit hydroquinone autoxidation. 4- To the extentthat comproportionation Of hydroquinone and quinone is the major propxgating pathway, superoxide dismutase will accelerate autoxidation. Acknow/ed~erfrrnu-We give special thanks to Enrique Cadenas. Gary Winston, and Christine Winterbourn discussions. , and REFERENCES I. Cadenas, E.; Mira. D.; Drumark, A,; Lind, C.; Segura-Aguilar. I: Ernster. L. Effect o f superoxide dismutase on the autoxidation o f various hydroquinones-a possible role o f superoxide dismutase as a superoxide:semiquinone oxidoreductase. Free Radical B i d . Med. 571-79; 1988. 2. Ollinper. K.; Buffington. G. D.; Ernster. L.;Cadenas, E. Effect o f superoxide dismutase on the autoxidation o f substituted hydro- and semi-naphthoquinones. Chem. B i d . lnreracr. 735376; 1990. 3. Winterbourn, C. C. Inhibition o f the autoxidation o f divicine and isouramil by the combination o f superoxide dismutase and reduced glutathione. Arch. Biochem. Biopltys. 2713447-455; 1989. 4. Ishii. T.: Fridovich, 1. Dual effects of superoxide dismutase on the autoxidation of 1,4-naphthohydroquinone. Free Radical Biol. Med. 8:21-24; 1990. 5 . Misra. H. P.; Fridovich, I . The role o f superoxide anion i n the autoxidation o f epinephrine and a simple assay for superoxide disniutase. J . B i d . Chem. 247:3170-3175; 1972. 6. Bandy, B.; Davison, A. I . Interactions between metals. ligands and oxygen i n the autoxidation o f 6-hydroxydopamine: mechanisms by which metal chelation enhances inhibition by superoxide dismutase. Arch. Biocltem. Biophys. 259:305-3 15; 1987. 7. Lovstad, R. A. Copper catalyzed oxidation o f ascorbate (vitamin C). 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DT-diaphorase-catalysed reduction o f I .4-naphthoquinone derivatives and glutathionyl-quinone conjugates. Effect of substituents on reaction rates. Biocherfr.J . 257561-57 I: 1989. 17. Marklund. S.; Marklund, G. Involvement o f superoxide anion radical in autoxidaticin o f pyrogallol and a convcnient :Issay for superoxide-disniutase. Eitr. 1. Bioc/irnr. 47:469-474: 1074 18. Moore. W. J . Phvsirnl rhrmistrv, 41h ed. Englewood CliTfs. NJ: Prentice-Hall Inc.; 1972:342. 19. Morton. R. A . Introductory account o f quinones. In: Morton. R. A.. ed. Biochemistry o f q i ~ i r i o t ~ eL.o~n.don: Acndeinic Press: 1965: 1-22.