Mitochondrial Aconitase Is a Source of Hydroxyl Radical

Abstract

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 ⅐ OH may be continuously generated in the mitochondria.There is much debate in the literature on the relative importance of hydroxyl radical ( ⅐ OH) and peroxynitrite in free radical pathology (1). Clarification of the mechanism centered on this subject is of considerable importance, especially in mitochondria, cellular organelles that are constantly exposed to low levels of superoxide anion (2, 3). Several neurodegenerative diseases (e.g. Alzheimer's disease, Parkinson's disease, and Lou Gehrig's disease or amyotrophic lateral sclerosis) and aging have been linked to mitochondrial oxidative damage that results in decreased mitochondrial function (3, 4). However, in biological systems, it is nearly impossible to associate a specific damage to a single oxidant. For example, superoxide and nitric oxide ( ⅐ NO) co-generated at very low levels (Ϸ10 Ϫ8 M) in cells will form peroxynitrite (ONOO Ϫ ) via a nearly diffusion-controlled reaction (5, 6). The toxicological significance of these species is clearly dependent on cell type, the biological targets, and their relationship to one another. One of the sensitive biological targets in oxidative damage to mitochondria is aconitase, an iron-sulfur protein that catalyzes the stereospecific dehydration-hydration of citrate to isocitrate in the Krebs cycle (7). Aconitase activity in mitochondria has been reported to be a sensitive redox sensor of reactive oxygen and nitrogen species in cells (8 -11). Aconitase contains a cubane-type [4Fe-4S] 2ϩ cluster in its active site with three iron atoms bound to cysteinyl groups and inorganic sulfur atoms and a fourth labile iron atom (Fe-␣). This Fe-␣ is unique in that it is not bound to a protein cysteine, but rather to a hydroxyl group of substrate and water (7 (13, 14). However, the reaction between aconitase and peroxynitrite is strongly inhibited by the addition of substrate that binds to the enzyme with high affinity (14).It was recently proposed that the reaction between mitochondrial aconitase (m-aconitase) 1 and superoxide plays a major role in mitochondrial oxidative damage (15)(16)(17). During this reaction, it has been proposed that iron is released from maconitase as iron(II) with the concomitant generation of hydrogen peroxide. This facilitates the formation of "free" hydroxyl radical in mitochondria. In the presence of intracellular reducing agents (e.g. glutathione, ascorbate, and NADPH), iron(II) is reincorporated into the inactive form of m-aconitase to regenerate the active form. According to this proposal, hydroxyl radical should be continuously generated in mitochondria as a result of the reaction between superoxide and aconitase. However, the experimental verification of this intriguing mechanism has so far been lacking.The objective of this study is to provide e...

Reversible redox-dependent modulation of mitochondrial aconitase and proteolytic activity during in vivo cardiac ischemia/reperfusion

“…Prooxidant-induced release of a labile ␣-Fe from the [4Fe-4S] 2ϩ cluster of aconitase results in loss in enzyme activity and in the formation of a [3Fe-4S] 1ϩ cluster that can be readily observed by EPR spectroscopy as a peak at g ϭ 2.02 with a shoulder at g ϭ 2.014 (10 K) (16,17,19). As shown in Fig.…”

“…Loss in aconitase activity is commonly used as a biomarker of oxidative damage due to the susceptibility of the enzyme's [4Fe-4S] 2ϩ cubane cluster to oxidative disassembly (16)(17)(18)(19). Nevertheless, we have recently shown that, when mitochondria are exposed to H 2 O 2 in vitro, aconitase can undergo oxidative inhibition followed by reactivation upon resolution of the oxidative stress (15).…”

“…Mitochondria, a major subcellular source of oxygen radicals during reperfusion, exhibit decrements in activity attributed, in part, to oxidative modifications to key metabolic enzymes (10). Aconitase and ␣-ketoglutarate dehydrogenase, which are known to be susceptible to oxidative inactivation in vitro (11)(12)(13)(14)(15)(16)(17)(18)(19) have been reported to decline in activity during in vivo cardiac ischemia͞ reperfusion (20).…”

Doxorubicin increases the susceptibility of brain mitochondria to Ca2+-induced permeability transition and oxidative damage

“…Mitochondrial aconitase contains a [4Fe-4S] 2+ cluster in its active site, which is oxidized by O 2 − and related species, generating the inactive [3Fe-4S] 1+ aconitase [52]. Moreover, O 2 − -mediated mitochondrial aconitase inactivation leads to hydroxyl radical formation [53].…”

Synoviocytes, not chondrocytes, release free radicals after cycles of anoxia/re-oxygenation

“…The superoxide anion that is produced on the matrix side of the mitochondrial membrane can cause cell damage (e.g., lipid peroxidation) by reacting with metal iron complexes within the enzymatic domain. Superoxide anion was also described as able to attack the aconitase, releasing the labile iron of the cluster [4Fe-4S] 2+ and forming Fe 2+ [10]. Superoxide anion also produces hydrogen peroxide (H 2 O 2 ) by subsequent dismutation, and both species, in the presence of Fe 2+ , may form the highly reactive species hydroxyl radicals which are known to cause cell damage especially by lipid peroxidation.…”