Advertisement

Mitochondrial Aconitase Is a Source of Hydroxyl Radical

AN ELECTRON SPIN RESONANCE INVESTIGATION*
Open AccessPublished:May 12, 2000DOI:https://doi.org/10.1074/jbc.275.19.14064
      Mitochondrial aconitase (m-aconitase) contains a [4Fe-4S]2+ cluster in its active site that catalyzes the stereospecific dehydration-rehydration of citrate to isocitrate in the Krebs cycle. It has been proposed that the [4Fe-4S]2+ aconitase is oxidized by superoxide, generating the inactive [3Fe-4S]1+ aconitase. In this reaction, the likely products are iron(II) and hydrogen peroxide. Consequently, the inactivation of m-aconitase by superoxide may increase the formation of hydroxyl radical (OH) through the Fenton reaction in mitochondria. In this work, evidence for the generation of OH from the reaction of m-aconitase with superoxide is provided using ESR spin trapping experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide and α-phenyl-N-tert-butylnitrone. Formation of free OH was verified with the OH scavenger Me2SO, which forms methyl radical upon reacting withOH. The addition of Me2SO to incubation mixtures containing m-aconitase and xanthine/xanthine oxidase yielded methyl radical, which was detected by ESR spin trapping. Methyl radical formation was further confirmed using [13C]Me2SO. Parallel low temperature ESR experiments demonstrated that the generation of the [3Fe-4S]1+ cluster increased with increasing additions of superoxide to m-aconitase. This reaction was reversible, as >90% of the initial aconitase activity was recovered upon treatment with glutathione and iron(II). This mechanism presents a scenario in whichOH may be continuously generated in the mitochondria.
      m-aconitase
      mitochondrial aconitase
      DEPMPO
      5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide
      DMPO
      5,5-dimethyl-1-pyrroline N-oxide
      PBN
      α-phenyl-N-tert-butylnitrone
      There is much debate in the literature on the relative importance of hydroxyl radical (OH) and peroxynitrite in free radical pathology (
      • Liochev S.I.
      • Fridovich I.
      ). Clarification of the mechanism centered on this subject is of considerable importance, especially in mitochondria, cellular organelles that are constantly exposed to low levels of superoxide anion (
      • Boveris A.
      • Chance B.
      ,
      • Wallace D.C.
      ). Several neurodegenerative diseases (e.g.Alzheimer's disease, Parkinson's disease, and Lou Gehrig's disease or amyotrophic lateral sclerosis) and aging have been linked to mitochondrial oxidative damage that results in decreased mitochondrial function (
      • Wallace D.C.
      ,
      • Dykens J.A.
      ). However, in biological systems, it is nearly impossible to associate a specific damage to a single oxidant. For example, superoxide and nitric oxide (NO) co-generated at very low levels (≈10−8m) in cells will form peroxynitrite (ONOO) via a nearly diffusion-controlled reaction (
      • Huie R.E.
      • Padjama S.
      ,
      • Kissner R.
      • Nauser T.
      • Bugnon P.
      • Lye P.G.
      • Koppenol W.
      ). The toxicological significance of these species is clearly dependent on cell type, the biological targets, and their relationship to one another. One of the sensitive biological targets in oxidative damage to mitochondria is aconitase, an iron-sulfur protein that catalyzes the stereospecific dehydration-hydration of citrate to isocitrate in the Krebs cycle (
      • Beinert H.
      • Kennedy M.C.
      • Stout C.D.
      ).
      Aconitase activity in mitochondria has been reported to be a sensitive redox sensor of reactive oxygen and nitrogen species in cells (
      • Gardner P.R.
      • Fridovich I.
      ,
      • Eisenstein R.S.
      • Kennedy M.C.
      • Beinert H.
      ,
      • Gardner P.R.
      • Fridovich I.
      ,
      • Gardner P.R.
      • Nguyen D.-D.H.
      • White C.W.
      ). Aconitase contains a cubane-type [4Fe-4S]2+ cluster in its active site with three iron atoms bound to cysteinyl groups and inorganic sulfur atoms and a fourth labile iron atom (Fe-α). This Fe-α is unique in that it is not bound to a protein cysteine, but rather to a hydroxyl group of substrate and water (
      • Beinert H.
      • Kennedy M.C.
      • Stout C.D.
      ). The labile Fe-α is released upon oxidation of the [4Fe-4S]2+cluster with the concomitant formation of inactive [3Fe-4S]1+ enzyme. Aconitase is inactivated rapidly by superoxide (k ≈ 107m−1 s−1) (
      • Flint D.H.
      • Tuminello J.F.
      • Emptage M.
      ) in the presence and absence of substrate and relatively slowly by peroxynitrite (k ≈ 105m−1s−1) and NO (
      • Castro L.
      • Rodriguez M.
      • Radi R.
      ,
      • Kennedy M.C.
      • Antholine W.E.
      • Beinert H.
      ). However, the reaction between aconitase and peroxynitrite is strongly inhibited by the addition of substrate that binds to the enzyme with high affinity (
      • Kennedy M.C.
      • Antholine W.E.
      • Beinert H.
      ).
      It was recently proposed that the reaction between mitochondrial aconitase (m-aconitase)1 and superoxide plays a major role in mitochondrial oxidative damage (
      • Fridovich I.
      ,
      • Gardner P.R.
      • Raineri I.
      • Epstein L.B.
      • White C.W.
      ,
      • Liochev S.I.
      ). During this reaction, it has been proposed that iron is released from m-aconitase as iron(II) with the concomitant generation of hydrogen peroxide. This facilitates the formation of “free” hydroxyl radical in mitochondria. In the presence of intracellular reducing agents (e.g. glutathione, ascorbate, and NADPH), iron(II) is reincorporated into the inactive form of m-aconitase to regenerate the active form. According to this proposal, hydroxyl radical should be continuously generated in mitochondria as a result of the reaction between superoxide and aconitase. However, the experimental verification of this intriguing mechanism has so far been lacking.
      The objective of this study is to provide evidence, using ESR, for the formation of hydroxyl radical and inactive [3Fe-4S]1+species from the reaction between superoxide and purified m-aconitase. Direct low temperature ESR was used to quantify [3Fe-4S]1+species.
      Note that [3Fe-4S]1+ is paramagnetic and hence ESR-active, whereas [4Fe-4S]2+ is diamagnetic and ESR-inactive.
      2Note that [3Fe-4S]1+ is paramagnetic and hence ESR-active, whereas [4Fe-4S]2+ is diamagnetic and ESR-inactive.
      Hydroxyl radical was detected by ESR spin trapping using a novel phosphorylated spin trap, 5-diethoxyphosphoryl-5-methylpyrroline N-oxide (DEPMPO), and a loop-gap resonator, which makes it possible to obtain ESR spectra using exceedingly small amounts of enzyme (
      • Kennedy M.C.
      • Emptage M.H.
      • Dreyer J.-L.
      • Beinert H.
      ). Our results indicate that the reaction between m-aconitase and superoxide releases iron(II) from the [4Fe-4S]2+ cluster, which can subsequently catalyze the formation of free hydroxyl radical. The biological implications of these reactions are discussed.

      Acknowledgments

      We thank Dr. William E. Antholine for assistance in low temperature ESR experiments.

      REFERENCES

        • Liochev S.I.
        • Fridovich I.
        Free Radical Biol. Med. 1999; 26: 777-778
        • Boveris A.
        • Chance B.
        Biochem. J. 1973; 156: 435-444
        • Wallace D.C.
        Science. 1999; 283: 1482-1488
        • Dykens J.A.
        Flint Beal M. Howell N. Bodis-Wollner I. Mitochondria and Free Radicals in Neurodegenerative Diseases. Wiley-Liss, New York1997: 29-55
        • Huie R.E.
        • Padjama S.
        Free Rad. Res. 1993; 18: 195-199
        • Kissner R.
        • Nauser T.
        • Bugnon P.
        • Lye P.G.
        • Koppenol W.
        Chem. Res. Toxicol. 1997; 10: 1285-1292
        • Beinert H.
        • Kennedy M.C.
        • Stout C.D.
        Chem. Rev. 1996; 96: 2335-2373
        • Gardner P.R.
        • Fridovich I.
        J. Biol. Chem. 1991; 266: 19328-19333
        • Eisenstein R.S.
        • Kennedy M.C.
        • Beinert H.
        Silver S. Walden W. Metal Ions in Gene Regulation. Chapman and Hill, Inc., New York1998: 157-216
        • Gardner P.R.
        • Fridovich I.
        J. Biol. Chem. 1992; 267: 8757-8763
        • Gardner P.R.
        • Nguyen D.-D.H.
        • White C.W.
        Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12248-12252
        • Flint D.H.
        • Tuminello J.F.
        • Emptage M.
        J. Biol. Chem. 1993; 268: 22369-22376
        • Castro L.
        • Rodriguez M.
        • Radi R.
        J. Biol. Chem. 1994; 269: 29409-29415
        • Kennedy M.C.
        • Antholine W.E.
        • Beinert H.
        J. Biol. Chem. 1997; 272: 20340-20347
        • Fridovich I.
        J. Biol. Chem. 1997; 272: 18515-18517
        • Gardner P.R.
        • Raineri I.
        • Epstein L.B.
        • White C.W.
        J. Biol. Chem. 1995; 270: 13399-13405
        • Liochev S.I.
        Free Radical Res. 1996; 25: 369-384
        • Vásquez-Vivar J.
        • Hogg N.
        • Martásek P.
        • Karoui H.
        • Tordo P.
        • Pritchard Jr., K.A.
        • Kalyanaraman B.
        Free Rad. Res. 1999; 31: 607-617
        • Kennedy M.C.
        • Emptage M.H.
        • Dreyer J.-L.
        • Beinert H.
        J. Biol. Chem. 1983; 258: 11098-11105
        • Ruzicka F.J.
        • Beinert H.
        J. Biol. Chem. 1978; 253: 2514-2517
        • Beinert H.
        • Emptage M.H.
        • Dreyer J.-L.
        • Scott R.A.
        • Han J.E.
        • Hodgson K.O.
        • Thomson A.J.
        Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 393-396
        • Flint D.H.
        • Allen R.M.
        Chem. Rev. 1996; 96: 2315-2334
        • Kennedy M.C.
        • Beinert H.
        J. Biol. Chem. 1988; 263: 8194-8198
        • Brazzolotto X.
        • Gaillard J.
        • Pantopoulos K.
        • Hentze M.W.
        • Moulis J.-M.
        J. Biol. Chem. 1999; 274: 21625-21630
        • Liochev S.I.
        • Fridovich I.
        Free Radical Biol. Med. 1994; 16: 29-33
        • Froncisz W.
        • Hyde J.S.
        J. Magn. Reson. 1982; 47: 515-521
        • Fréjaville C.
        • Karoui H.
        • Tuccio B.
        • LeMoigne F.
        • Culcasi M.
        • Pietri S.
        • Lauricella R.
        • Tordo P.
        J. Med. Chem. 1995; 38: 258-265
        • Konorev E.A.
        • Kennedy M.C.
        • Kalyanaraman B.
        Arch. Biochem. Biophys. 1999; 368: 421-428
        • Minotti G.
        • Aust S.D.
        Free Radical Biol. Med. 1987; 3: 379-387
        • Vile G.F.
        • Winterbourn C.C.
        Biochem. Pharmacol. 1988; 37: 2893-2897
        • Mello-Filho A.C.
        • Meneghini R.
        Mutat. Res. 1991; 251: 109-113