Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide.
Mitochondrial and cytosolic aconitases have been indicated as major targets of .NO- and O2-.-mediated toxicity in cells due to the oxidant-mediated disruption of the [4Fe-4S] prosthetic group. However, under circumstances in which both .NO and O2-. are generated, their almost diffusion-controlled combination reaction (k = 6.7 x 10(9) M-1 s-1), leading to the formation of peroxynitrite anion (ONOO-), can out-compete the direct reactions of .NO and O2-. with aconitase and even the enzymatic dismutation of O2-. by superoxide dismutase. In this work, we report that ONOO- reacts with isolated pig heart mitochondrial aconitase at 1.4 x 10(5) M-1 s-1, resulting in a significant loss of enzymatic activity. Aconitase activity was totally recovered after postincubation with thiols and ferrous iron, indicating that ONOO- reactions with the enzyme involve the perturbation of the labile Fe alpha to yield the inactive [3Fe-4S] cluster, which is also evident by spectral changes. On the other hand, anaerobic exposure of isolated aconitase to high concentrations of .NO (> 100 microM) led to a moderate inhibition of the enzyme, which could be fully overcome by .NO displacement under an argon-saturated atmosphere, in agreement with the formation of a reversible inhibitory complex between .NO and the active site of aconitase. Superoxide inactivated mitochondrial aconitase at (3.5 +/- 2) x 10(6) M-1 s-1, a reaction rate 3 orders of magnitude slower than its reaction rate with .NO. O2-. could represent the main mechanism of inactivation of the enzyme in systems in which it is formed without significant concomitant production of .NO. Our results imply that the mechanisms by which .NO and O2-. inactivate aconitase in cell systems may not be simple due to their direct reactions with the iron-sulfur cluster, but may rely on the formation of ONOO-.
Department of Biochemistry, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
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D000154:Aconitate Hydratase / Q000037:antagonists & inhibitors*; D000818:Animals; D007506:Iron-Sulfur Proteins / Q000187:drug effects; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology; D009566:Nitrates / Q000494:pharmacology*; D009569:Nitric Oxide / Q000494:pharmacology*; D009603:Nitroso Compounds / Q000494:pharmacology; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013552:Swine
D007506:Iron-Sulfur Proteins; D009566:Nitrates; D009603:Nitroso Compounds; D013438:Sulfhydryl Compounds; C039642:peroxynitric acid; D009569:Nitric Oxide; D000154:Aconitate Hydratase