Mitochondrial aconitase is a source of hydroxyl radical. An electron spin resonance investigation.
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 alpha-phenyl-N-tert-butylnitrone. Formation of free ( small middle dot)OH was verified with the (*)OH scavenger Me(2)SO, which forms methyl radical upon reacting with (*)OH. The addition of Me(2)SO 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 [(13)C]Me(2)SO. 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 which (*)OH may be continuously generated in the mitochondria.
Vasquez-Vivar
J
J
Department of Pathology, Cardiovascular Research Center, the Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA. jvvivar@mcw.edu
Kalyanaraman
B
B
Kennedy
M C
MC
D000154:Aconitate Hydratase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002384:Catalysis; D002417:Cattle; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D017665:Hydroxyl Radical / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis; D008929:Mitochondria, Heart / Q000201:enzymology*; D013113:Spin Labels; D013481:Superoxides / Q000378:metabolism
D003497:Cyclic N-Oxides; D007506:Iron-Sulfur Proteins; D013113:Spin Labels; D013481:Superoxides; C098505:5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide; D017665:Hydroxyl Radical; D000154:Aconitate Hydratase