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Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide
L Castro1, M Rodriguez, R Radi
1Department of Biochemistry, Facultad de Medicina, Universidad de la República, Montevideo, Uruguay.
Abstract:
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-.
Insights
Peroxynitrite (ONOO-) inactivates mitochondrial aconitase by disrupting its iron-sulfur cluster, while nitric oxide (.NO) causes reversible inhibition and superoxide (O2-.) causes slower inactivation. ONOO- formation is key in combined .NO and O2-. environments.
Area of Science:
- Biochemistry
- Cellular Biology
- Enzymology
Background:
- Mitochondrial and cytosolic aconitases are susceptible to nitric oxide (.NO) and superoxide (O2-.) toxicity due to oxidant-mediated disruption of their [4Fe-4S] prosthetic groups.
- In biological systems generating both .NO and O2-., their rapid combination forms peroxynitrite anion (ONOO-), potentially outcompeting direct reactions with aconitase or superoxide dismutase.
- Understanding the specific inactivation mechanisms of aconitase by these reactive species is crucial for cellular redox homeostasis.
Purpose of the Study:
- To investigate the direct reaction kinetics and mechanisms of peroxynitrite anion (ONOO-), nitric oxide (.NO), and superoxide (O2-.) with isolated pig heart mitochondrial aconitase.
- To elucidate the role of ONOO- formation in aconitase inactivation when both .NO and O2-. are present.
Main Methods:
- Enzymatic activity assays of isolated pig heart mitochondrial aconitase.
- Kinetic analysis of aconitase inactivation by ONOO-, .NO, and O2-.
- Spectroscopic analysis to characterize changes in the aconitase iron-sulfur cluster.
- Reversal studies using thiols and ferrous iron to assess the nature of inactivation.
Main Results:
- Peroxynitrite (ONOO-) rapidly inactivates mitochondrial aconitase (k = 1.4 x 10^5 M^-1 s^-1), leading to loss of activity via perturbation of the labile Fe alpha and formation of an inactive [3Fe-4S] cluster.
- Nitric oxide (.NO) causes moderate, reversible inhibition of aconitase at high concentrations, suggesting binding to the active site.
- Superoxide (O2-.) inactivates aconitase more slowly (k = 3.5 x 10^6 M^-1 s^-1) than .NO, but significantly slower than ONOO-.
Conclusions:
- Peroxynitrite (ONOO-) is a potent inactivator of mitochondrial aconitase, primarily through disruption of its iron-sulfur cluster.
- The formation of ONOO- is likely the dominant mechanism for aconitase inactivation in cellular environments where both .NO and O2-. are concurrently produced.
- Direct reaction with .NO results in reversible inhibition, while O2-. inactivation is slower, highlighting distinct inactivation pathways.