Mitochondrial aconitase is a source of hydroxyl radical - An electron spinresonance investigation

Mitochondrial aconitase (m-aconitase) contains a [4Fe-4S](2+) cluster in it s active site that catalyzes the stereospecific dehydration-rehydration of citrate to isocitrate in the Krebs cycle. It has been proposed that the [4F e-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 superox ide may increase the formation of hydroxyl radical ((OH)-O-.) through the F enton reaction in mitochondria. In this work, evidence for the generation o f (OH)-O-. from the reaction of m-aconitase with superoxide is provided usi ng ESR spin trapping experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrro line N-oxide and alpha-phenyl-N-tert-butylnitrone. Formation of free (OH)-O -. was verified with the (OH)-O-. scavenger Me2SO, which forms methyl radic al upon reacting with (OH)-O-.. The addition of Me2SO to incubation mixture s containing m-aconitase and xanthine/xanthine oxidase yielded methyl radic al, which was detected by ESR spin trapping. Methyl radical formation was f urther confirmed using [C-13]Me2SO. Parallel low temperature ESR experiment s demonstrated that the generation of the [3Fe-4S](1+) cluster increased wi th increasing additions of superoxide to m-aconitase. This reaction was rev ersible, as >90% of the initial aconitase activity was recovered upon treat ment with glutathione and iron(II). This mechanism presents a scenario in w hich (OH)-O-. may be continuously generated in the mitochondria.