Nitric Oxide Sensitivity of the Aconitases

Abstract

Aconitases are important cellular targets of nitric oxide (NO⋅) toxicity, and NO⋅-derived species, rather than NO⋅ per se, have been proposed to mediate their inactivation. NO⋅-mediated inactivation of theEscherichia coli aconitase and the porcine mitochondrial aconitase was investigated. In E. coli, aconitase activity decreased by ∼70% during a 2-h exposure to an atmosphere containing 120 ppm NO⋅ in N2. The NO⋅-inactivated aconitase reactivated poorly in E. coli under anaerobic or aerobic conditions. Elevated superoxide dismutase activity did not affect the aerobic inactivation of aconitase by NO⋅, thus indicating a limited role of the NO⋅- and superoxide-derived species peroxynitrite. Glutathione-deficient and glutathione-containingE. coli were comparably sensitive to NO⋅-mediated aconitase inactivation, thus excluding the participation of S-nitrosoglutathione or more oxidizing NO⋅-derived species. NO⋅ progressively decreased aconitase activity in extracts in the presence of substrates, and inactivation was greatest at an acidic pH withcis-aconitate. The porcine mitochondrial aconitase was sensitive to NO⋅ when exposed at pH 6.5, but not at pH 7.5, and irreversible inactivation occurred during catalysis. The requirement of an acidic pH or substrates for sensitivity may explain the reported resistance of aconitases to NO⋅ in vitro (Castro, L., Rodriguez, M., and Radi, R. (1994) J. Biol. Chem.269, 29409–29415; Hausladen, A., and Fridovich, I. (1994)J. Biol. Chem. 269, 29405–29408). AnS-nitrosation of the aconitase [4Fe-4S] center catalyzed by the solvent-exposed electron withdrawing iron atom (Fea) is proposed. Aconitases are important cellular targets of nitric oxide (NO⋅) toxicity, and NO⋅-derived species, rather than NO⋅ per se, have been proposed to mediate their inactivation. NO⋅-mediated inactivation of theEscherichia coli aconitase and the porcine mitochondrial aconitase was investigated. In E. coli, aconitase activity decreased by ∼70% during a 2-h exposure to an atmosphere containing 120 ppm NO⋅ in N2. The NO⋅-inactivated aconitase reactivated poorly in E. coli under anaerobic or aerobic conditions. Elevated superoxide dismutase activity did not affect the aerobic inactivation of aconitase by NO⋅, thus indicating a limited role of the NO⋅- and superoxide-derived species peroxynitrite. Glutathione-deficient and glutathione-containingE. coli were comparably sensitive to NO⋅-mediated aconitase inactivation, thus excluding the participation of S-nitrosoglutathione or more oxidizing NO⋅-derived species. NO⋅ progressively decreased aconitase activity in extracts in the presence of substrates, and inactivation was greatest at an acidic pH withcis-aconitate. The porcine mitochondrial aconitase was sensitive to NO⋅ when exposed at pH 6.5, but not at pH 7.5, and irreversible inactivation occurred during catalysis. The requirement of an acidic pH or substrates for sensitivity may explain the reported resistance of aconitases to NO⋅ in vitro (Castro, L., Rodriguez, M., and Radi, R. (1994) J. Biol. Chem.269, 29409–29415; Hausladen, A., and Fridovich, I. (1994)J. Biol. Chem. 269, 29405–29408). AnS-nitrosation of the aconitase [4Fe-4S] center catalyzed by the solvent-exposed electron withdrawing iron atom (Fea) is proposed. Nitric oxide (NO⋅) is released as an intermediate of dissimilatory and assimilatory pathways of nitrite reduction in bacteria and fungi (1Goretski J. Zafiriou O.C. Hollocher T.C. J. Biol. Chem. 1990; 265: 11535-11538Abstract Full Text PDF PubMed Google Scholar, 2Ji X. Hollocher T.C. Biochem. Biophys. Res. Commun. 1988; 157: 106-108Crossref PubMed Scopus (74) Google Scholar). It is also produced by macrophages, neutrophils, endothelial cells, and epithelial cells to combat invading microorganisms and neoplastic tissue (3Nathan C. Hibbs Jr., J.B. Curr. Opin. Immunol. 1991; 3: 65-70Crossref PubMed Scopus (1322) Google Scholar, 4De Groote, M. A., and Fang, F. C. (1995) Clin. Infect. Dis. , 21, Suppl. 2, 162–165Google Scholar, 5Drapier J.-C. Hibbs J.B. J. Immunol. 1994; 14: 2829-2838Google Scholar). In addition, NO⋅serves important functions as a second messenger for neurons and the vascular system (6Garthwaite J. Boulton C.L. Annu. Rev. Physiol. 1995; 57: 683-706Crossref PubMed Scopus (1533) Google Scholar, 7Hobbs A.J. Ignarro L.J. Methods Enzymol. 1996; 269: 134-148Crossref PubMed Google Scholar). NO⋅ is cytotoxic. Mechanisms of NO⋅ toxicity are complex and may include its direct reaction with heme, non-heme iron, and copper proteins (8Henry Y. Lepoivre M. Drapier J.-C. Ducrocq C. Boucher J.-L. Guissani A. FASEB J. 1993; 7: 1124-1134Crossref PubMed Scopus (352) Google Scholar). Other NO⋅-derived species including nitroxyl anion, nitrosonium, peroxynitrite (ONOO−), nitrogen dioxide, and nitrosothiols may increase the spectrum of NO⋅-mediated damage to cells (9Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Crossref PubMed Scopus (2441) Google Scholar). The avid reactivity of NO⋅ with superoxide radical (O·̄2) to form the less discriminant oxidant ONOO− provides a potent mechanism for the deleterious actions of NO⋅ (10Pryor W.A. Squadrito G.L. Am. J. Physiol. 1995; 268: L699-L722PubMed Google Scholar, 11Czapski G. Goldstein S. Free Radical Biol. Med. 1995; 19: 785-794Crossref PubMed Scopus (129) Google Scholar). Nevertheless, a thorough understanding of NO⋅ toxicity continues to demand a greater knowledge of the NO⋅-sensitive targets and the NO⋅-derived species involved. The mammalian [4Fe-4S] cytoplasmic and mitochondrial aconitases are particularly sensitive to inactivation by NO⋅ produced during various pathological conditions (12Drapier J.-C. Hibbs Jr, J.B. Methods Enzymol. 1996; 269: 26-36Crossref PubMed Google Scholar, 13Bouton C. Raveau M. Drapier J.C. J. Biol. Chem. 1996; 271: 2300-2306Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 14Rouault T.A. Klausner R.D. Trends Biochem. Sci. 1996; 21: 174-177Abstract Full Text PDF PubMed Scopus (231) Google Scholar, 15Drapier J.-C. Hibbs J.B. J. Clin. Invest. 1986; 78: 790-797Crossref PubMed Scopus (368) Google Scholar, 16Stadler J. Billiar T.R. Curran R.D. Stuehr D.J. Ochoa J.B. Simmons R.L. Am. J. Physiol. 1991; 260: C910-C916Crossref PubMed Google Scholar, 17Stadler J. Curran R.D. Ochoa J.B. Harbrecht B.G. Hoffman R.A. Simmons R.L. Billiar T.R. Arch. Surg. 1994; 126: 186-191Crossref Scopus (111) Google Scholar, 18Drapier J.-C. Hirling H. Wietzerbin J. Kaldy P. Kühn L.C. EMBO J. 1993; 12: 3643-3649Crossref PubMed Scopus (345) Google Scholar). In mammals, the cytoplasmic aconitase serves as an mRNA-binding regulator of iron homeostasis and may also participate with the mitochondrial aconitase as a catalyst of the energy-yielding reactions of the citric acid cycle (19Klausner R.D. Rouault T.A. Harford J.B. Cell. 1993; 72: 19-28Abstract Full Text PDF PubMed Scopus (1051) Google Scholar, 20Philpott C.C. Klausner R.D. Rouault T.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7321-7325Crossref PubMed Scopus (117) Google Scholar). In addition, there is a growing family of homologous labile [4Fe-4S] (de)hydratases serving important biosynthetic and energy-yielding functions (21Gardner P.R. Biosci. Rep. 1997; 17: 33-42Crossref PubMed Scopus (151) Google Scholar, 22Flint D.H. Chem. Rev. 1996; 96: 2315-2334Crossref PubMed Scopus (220) Google Scholar), which may account for the toxicity of NO⋅toward a variety of organisms under a variety of conditions. Yet, it remains unclear how NO⋅ inactivates aconitases in vivo. The NO⋅ resistance of various isolated aconitases has suggested the involvement of more reactive NO⋅-derived species, such as ONOO− or the nitrosothiol GSNO 1The abbreviations used are: GSNO,S-nitrosoglutathione; LB, Luria broth; SOD, superoxide dismutase; Mn-SOD, manganese-containing SOD; Cu,Zn-SOD, copper- and zinc-containing SOD; Mes, 4-morpholineethanesulfonic acid.1The abbreviations used are: GSNO,S-nitrosoglutathione; LB, Luria broth; SOD, superoxide dismutase; Mn-SOD, manganese-containing SOD; Cu,Zn-SOD, copper- and zinc-containing SOD; Mes, 4-morpholineethanesulfonic acid. (23Hausladen A. Fridovich I. J. Biol. Chem. 1994; 269: 29405-29408Abstract Full Text PDF PubMed Google Scholar, 24Castro L. Rodriguez M. Radi R. J. Biol. Chem. 1994; 269: 29409-29415Abstract Full Text PDF PubMed Google Scholar). Here we describe the NO⋅-mediated inactivation of aconitase inEscherichia coli that occurs independently of ONOO− or GSNO formation and produces a poorly reactivatable aconitase. We also describe the effects of pH and substrates on the reaction of NO⋅ with the E. coliand mitochondrial aconitases in vitro. The results may account for the apparent NO⋅ resistance of aconitases (23Hausladen A. Fridovich I. J. Biol. Chem. 1994; 269: 29405-29408Abstract Full Text PDF PubMed Google Scholar, 24Castro L. Rodriguez M. Radi R. J. Biol. Chem. 1994; 269: 29409-29415Abstract Full Text PDF PubMed Google Scholar). Nitrosation of the aconitase [4Fe-4S] center catalyzed by the solvent-exposed iron atom (Fea) is proposed. E. coli strain DH5α was from Life Technologies. The E. colimutant JTG10 (gshA−), lacking glutathione, and its parental strain AB1157 were kindly provided by B. Demple (25Greenberg J.T. Demple B. J. Bacteriol. 1986; 168: 1026-1029Crossref PubMed Google Scholar). DH5α was transformed with the multicopy plasmid pD11c (26Gardner P.R. Fridovich I. J. Biol. Chem. 1993; 268: 12958-12963Abstract Full Text PDF PubMed Google Scholar) containing the E. coli sodA gene, and cultures of DH5αpD11c were maintained with 100 μg/ml ampicillin. The porcine mitochondrial aconitase expression vector pA was generously provided by H. Zalkin (27Zheng L. Kennedy M.C. Beinert H. Zalkin H. J. Biol. Chem. 1992; 267: 7895-7903Abstract Full Text PDF PubMed Google Scholar) and was introduced into E. coli strain BL21(DE3)LysS by standard methods. 1200 ppm (± 5%) NO⋅ in ultrapure nitrogen, 99.998% N2, and 99.993% O2 compressed gas cylinders were obtained from Praxair (Bethlehem, PA). Nitric oxide (98.5%) was from Aldrich. Porcine heart isocitrate dehydrogenase, barium (±)-fluorocitrate, MnCl2, sodium cis-aconitate, sodium citrate, sodium trans-aconitate, sodium tricarballylate, NADP+, horse heart cytochrome c 3+, xanthine, xanthine oxidase, phenazine methosulfate, porcine heart mitochondrial aconitase, chloramphenicol, bovine erythrocyte Cu,Zn-SOD (3500 units/mg), succinic acid, bovine serum albumin (fraction V), dithiothreitol, ferrous ammonium sulfate, Sephadex G25, reactive blue 2-Sepharose, Mes, Tris, Hepes, and KCl were from Sigma. Bacto-tryptone and yeast extract were obtained from Difco. The minimal salts medium was made up with tap water and contained 60 mm K2HPO4, 33 mmKH2PO4, 7.6 mm(NH4)2SO4, 1.7 mmsodium citrate, 1 mm MgSO4, 10 μmMnCl2, 10 μg/ml thiamine-Cl, 40 μg/mll-arginine-Cl, and 10 mm sodium succinate. The 20 common l-amino acids were supplied at 40 μg/ml as indicated with cystine instead of cysteine. The phosphate-buffered LB medium (28Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Scholar) contained 10 of of yeast and 10 of per of 33 10 The pH of the LB and minimal salts was to with or were in a water at with a of at and was at the of were with a a and to the were to and were at for the was and the was on were and by the in of a containing mm pH mm MnCl2, and 20 (±)-fluorocitrate, and the was on The barium of was by a with was to aconitase in extracts P.R. Fridovich I. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). of did not affect of aconitase activity in and were at for up to of aconitase were by in a water and by for at to the of aconitase the of a of with a from an in minimal salts was to an at of in minimal salts were by at for at were and in of 100 pH 7.5, by with a were by at and were at activity was in a reaction containing mm pH mm sodium citrate, mm and 1 isocitrate by the increase at at for P.R. Fridovich I. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). of aconitase activity to 1 of per was by the of Biochem. 72: PubMed Scopus Google Scholar) and bovine serum albumin as the activity was by the of cytochrome c reduction Fridovich I. J. Biol. Chem. Full Text PDF PubMed Google Scholar) in a at and were from a standard with bovine Cu,Zn-SOD (3500 A Molecular was to the of aconitase, SOD, and A with a was used for A gas was used to and a of for various of N2, and NO⋅, and gas were a containing sodium to and of nitrogen to reaction The was for by the of a of at and NO⋅ were by water with NO⋅ that was NO⋅ was into containing aconitase in a which for in gas It was important to gas to NO⋅ in the Porcine mitochondrial aconitase was produced and isolated from E. coli strain with to (27Zheng L. Kennedy M.C. Beinert H. Zalkin H. J. Biol. Chem. 1992; 267: 7895-7903Abstract Full Text PDF PubMed Google Scholar). aconitase of and of the as from was reactivated in a reaction containing mm ferrous ammonium and mm with 100 mm pH 7.5, under at for M.C. J.-L. Beinert H. J. Biol. Chem. 258: Full Text PDF PubMed Google Scholar). aconitase was on Sephadex with 100 mm pH the of the aconitase at pH 6.5, aconitase was in 100 mm pH of aconitase were under at in are of or more The sensitivity of the aconitase activity coli to NO⋅ was by cultures to a atmosphere containing NO⋅ with N2. activity was during exposure of cultures to 120 ppm NO⋅ gas aconitase activity was during under were in the presence of the to to The results a sensitivity of the E. coli aconitase to of NO⋅ and that NO⋅-derived species, such as are not for aconitase inactivation. The aconitase center is in a of in E. coli and T.A. Klausner R.D. Trends Biochem. Sci. 1996; 21: 174-177Abstract Full Text PDF PubMed Scopus (231) Google Scholar, P.R. Biosci. Rep. 1997; 17: 33-42Crossref PubMed Scopus (151) Google P.R. Fridovich I. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, P.R. I. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). In the solvent-exposed and released by in aconitase activity is by reduction of the center and coli, of the aconitase occurs with a of P.R. Fridovich I. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). We the of NO⋅-inactivated aconitase of cultures to an atmosphere containing ppm NO⋅ in for 60 of aconitase activity 2, of NO⋅ and with nitrogen the exposure a of activity of the activity the presence of O2 the of the NO⋅-inactivated aconitase of the activity was for the of the activity was aconitase activity in E. coli was under or during the from to O2 It that of aconitase activity was when aconitase was by P.R. Fridovich I. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, P.R. I. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). The and of the NO⋅-inactivated aconitase a mechanism of inactivation a less by NO⋅ than that with Aconitases are by ONOO− and GSNO in vitro (23Hausladen A. Fridovich I. J. Biol. Chem. 1994; 269: 29405-29408Abstract Full Text PDF PubMed Google Scholar, 24Castro L. Rodriguez M. Radi R. J. Biol. Chem. 1994; 269: 29409-29415Abstract Full Text PDF PubMed Google Scholar), and for ONOO− or GSNO have been proposed to explain the NO⋅-mediated of aconitase activity in vivo. We the effects of and on the NO⋅-mediated inactivation of aconitase in E. coli to the participation of ONOO− and GSNO in aconitase inactivation. E. coli strain DH5αpD11c the was with its parental strain DH5α for its sensitivity to NO⋅-mediated aconitase inactivation inactivation during NO⋅ exposure was in DH5α and DH5αpD11c 1 and aconitase activity was during to O2 in DH5αpD11c and in DH5α activity was more to the phenazine in DH5αpD11c than in DH5α as for a of activity P.R. Fridovich I. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). The results a limited role of and ONOO− in the mechanism of NO⋅-mediated aconitase inactivation. The E. coli lacking glutathione, and its parental strain AB1157 (25Greenberg J.T. Demple B. J. Bacteriol. 1986; 168: 1026-1029Crossref PubMed Google Scholar) were used to the effects of cellular on NO⋅-mediated aconitase inactivation. The of did not affect the NO⋅-mediated of aconitase activity in strain JTG10 to AB1157 under aerobic conditions 1 and or anaerobic conditions 1 and It that JTG10 contained a of aconitase and more than AB1157 under aerobic conditions as reported P.R. Fridovich I. Arch. Biochem. Biophys. 1993; PubMed Scopus Google the of activity during was in The of to aconitase a mechanism of inactivation of the of in E. coli P. Biophys. PubMed Scopus Google Scholar) to oxidizing species such as ONOO− R. J.S. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). the results not a of NO⋅ H. T. J. J.S. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) or the participation of GSNO in the mechanism of aconitase inactivation. We the reaction of the aconitases in vitro. was in extracts exposed to 1200 ppm NO⋅ in and the substrates trans-aconitate, and P.R. Fridovich I. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, Kennedy M.C. Beinert H. Hoffman 1990; PubMed Scopus Google Scholar, J. H. Kennedy M.C. Beinert H. Hoffman J. Biol. Chem. 1986; Full Text PDF PubMed Google Scholar) inactivation with 1 In addition, aconitase activity was decreased by NO⋅ exposure during with the P.R. Fridovich I. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar) the with mm KCl the of NO⋅ and the pH of the reaction from to the inactivation with and the E. coli aconitase was more by NO⋅ during with the than with and The results a mechanism the reaction of NO⋅ or an NO⋅-derived species, with the [4Fe-4S] center and that the NO⋅-mediated inactivation in occurs with the is by acidic and NO⋅ to more the of E. coli aconitase by NO⋅ during catalysis. of E. to a reaction under at and containing or mm mm MnCl2, 1 isocitrate dehydrogenase, and 1 bovine serum were with mm pH 1 and or mm Hepes, pH and and were exposed under a of 1200 ppm NO⋅ N2, or in and were at to gas were in the presence of mm were with a and for at The of aconitase activity was from the of increase of at under NO⋅ to that for reactions under N2. were with 40 1 and and 100 of extract The results did not the involvement of a NO⋅-derived species in the mechanism of aconitase inactivation, we were extracts the effects of and substrates on the inactivation of the E. coli aconitase provided a for understanding the of of NO⋅ to aconitases in vitro (23Hausladen A. Fridovich I. J. Biol. Chem. 1994; 269: 29405-29408Abstract Full Text PDF PubMed Google Scholar, 24Castro L. Rodriguez M. Radi R. J. Biol. Chem. 1994; 269: 29409-29415Abstract Full Text PDF PubMed Google Scholar). we that acid pH or may the direct inactivation of aconitases by NO⋅ and that the NO⋅ sensitivity of the porcine heart mitochondrial aconitase may also more under conditions. with of the porcine mitochondrial aconitase with NO⋅ at pH to aconitase during a exposure 1 and L. Rodriguez M. Radi R. J. Biol. Chem. 1994; 269: 29409-29415Abstract Full Text PDF PubMed Google Scholar). an apparent of aconitase activity at pH in the presence 1 and or and of mm at pH 6.5, exposure of the aconitase to an atmosphere of 1200 ppm NO⋅ a of aconitase activity to the 1 and the activity was by the A NO⋅ resistance was with the porcine mitochondrial aconitase at pH and NO⋅-mediated inactivation was at pH during exposure of the to 100 the E. coli aconitase, the of porcine mitochondrial aconitase and the isolated were by NO⋅ during with of at pH and The was not by the reaction with to NO⋅ indicated by the A of was with at pH and 1 and the of KCl the resistance of the mitochondrial aconitase to NO⋅ and with 1 We that NO⋅ and with the aconitases and that inactivation occurs with the of porcine mitochondrial aconitase by NO⋅ during catalysis. Porcine mitochondrial aconitase in a reaction with at pH A and or pH and and containing mm cis-aconitate, mm MnCl2, 1 isocitrate dehydrogenase, and 1 bovine serum albumin was exposed to 1 and or 1200 ppm NO⋅ in and in the presence 1 and or and of mm KCl at porcine heart aconitase A or porcine mitochondrial aconitase and were at and were exposed under a gas in and were on a water at to gas were with a and for at The the of NO⋅ in with of NO⋅ gas exposure the aconitase in E. We the of NO⋅ for aconitase inactivation to at an NO⋅ gas of 120 ppm we a of NO⋅ in the bacteria to that in and the NO⋅ for water of mm at 1 atmosphere and 20 A of the NO⋅-inactivated aconitase to to the NO⋅ sensitivity of the aconitase in vivo. a inactivation of the aconitase by NO⋅ with a may have effects with of the inactivation and with the of and aconitase in by the of inactivation and (21Gardner P.R. Biosci. Rep. 1997; 17: 33-42Crossref PubMed Scopus (151) Google Scholar, P.R. Fridovich I. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). or more oxidizing NO⋅-derived species not to in the mechanism of aconitase inactivation coli as by the of or to affect the NO⋅-mediated inactivation and by the of inactivation. O2 and may aconitase by the of NO⋅ the formation of ONOO− and ONOO− to form at a in E. coli under aerobic conditions and to the aconitase we were to the of of by E. Fridovich I. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar), the of reaction NO⋅ and of S. Free Radical Res. Commun. 1993; PubMed Scopus Google Scholar), and the second for the reaction of ONOO− with aconitase of L. Rodriguez M. Radi R. J. Biol. Chem. 1994; 269: 29409-29415Abstract Full Text PDF PubMed Google Scholar). the of ONOO− by various or the of substrates to aconitase may the of the aconitase center in vivo. In NO⋅ may have a and reactivity with the solvent-exposed of the aconitase center to that of It in that NO⋅ J.-C. Hirling H. Wietzerbin J. Kaldy P. Kühn L.C. EMBO J. 1993; 12: 3643-3649Crossref PubMed Scopus (345) Google Scholar) and NO⋅ C. Raveau M. Drapier J.C. J. Biol. Chem. 1996; 271: 2300-2306Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) were reported to the mammalian cytoplasmic aconitase in extracts independently of and thus ONOO− in the of NO⋅ the inactivation of aconitase a was not aconitase was by NO⋅ in vitro. The aconitase was more than the aconitase an of on the reactivity of NO⋅ with the solvent-exposed to may the NO⋅ or may increase the reactivity by the of The the in the mitochondrial aconitase as indicated by and Kennedy M.C. Beinert H. Hoffman 1990; PubMed Scopus Google Scholar, J. H. Kennedy M.C. Beinert H. Hoffman J. Biol. Chem. 1986; Full Text PDF PubMed Google Scholar, T.A. Kennedy M.C. Beinert H. E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). the of the and to aconitase with that of the that an intermediate in the of aconitase or a of the aconitase the reactivity of NO⋅ rather than to the isolated mitochondrial aconitase was to inactivation by NO⋅ at pH of in which the isolated and E. coli aconitases were to to NO⋅ at the of at pH 7.6 or (23Hausladen A. Fridovich I. J. Biol. Chem. 1994; 269: 29405-29408Abstract Full Text PDF PubMed Google Scholar, 24Castro L. Rodriguez M. Radi R. J. Biol. Chem. 1994; 269: 29409-29415Abstract Full Text PDF PubMed Google Scholar). we were to that the mitochondrial aconitase is at a more acidic pH during the exposure to of 1200 ppm NO⋅ or with of NO⋅ in the reactivity of NO⋅ may by the effects of pH on to the solvent-exposed of the aconitase Kennedy M.C. Beinert H. Hoffman 1990; PubMed Scopus Google Scholar, B. A.J. J. Biochem. 1995; PubMed Scopus Google Scholar) or may to effects on acid in the (27Zheng L. Kennedy M.C. Beinert H. Zalkin H. J. Biol. Chem. 1992; 267: 7895-7903Abstract Full Text PDF PubMed Google Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). NO⋅ has an for and non-heme iron, and it that NO⋅ to of the aconitase the of we have of of NO⋅ with the center of the mitochondrial aconitase that with the of activity M.C. Beinert H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the acid of to from NO⋅ to an intermediate with or to in the In the reaction of NO⋅ at the of aconitase may to the of albumin catalyzed by M. R. A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) with the that the reaction and the electron withdrawing of such as and substrates, may formation and A of the center may also explain the of the aconitase in It to the NO⋅ sensitivity of aconitase to of the [4Fe-4S] family or to it important to the NO⋅-mediated to the that its it is that the NO⋅-sensitive aconitases may into the of NO⋅ toxicity and the the of the aconitases may increase the of functions to the NO⋅- and conditions of the during H. C.C. Am. J. Physiol. 1994; Google Scholar) or of the during the to Rev. Infect. Dis. PubMed Scopus Google Scholar). We of the for and We with Zalkin for kindly the porcine aconitase expression plasmid and Demple for E. We are to Beinert and for a of of the of NO⋅ with the mitochondrial aconitase.