Sites and Mechanisms of Aconitase Inactivation by Peroxynitrite:  Modulation by Citrate and Glutathione

Abstract

Aconitases are iron-sulfur cluster-containing proteins present both in mitochondria and cytosol of cells; the cubane iron-sulfur (Fe-S) cluster in the active site is essential for catalytic activity, but it also renders aconitase highly vulnerable to reactive oxygen and nitrogen species. This study examined the sites and mechanisms of aconitase inactivation by peroxynitrite (ONOO-), a strong oxidant and nitrating agent readily formed from superoxide anion and nitric oxide generated by mitochondria. ONOO- inactivated aconitase in a dose-dependent manner (half-maximal inhibition was observed with approximately 3 microM ONOO-). Low levels of ONOO- caused the conversion of the Fe-S cluster from the [4Fe-4S]2+ form to the inactive [3Fe-4S]1+ form with the loss of labile iron, as confirmed by low-temperature EPR analysis. In the presence of the substrate, citrate, 66-fold higher concentrations of ONOO- were required for half-maximal inhibition. The protective effects of citrate corresponded to its binding to the active site. The inactivation of aconitase in the presence of citrate was due to ONOO--mediated cysteine thiol loss and tyrosine nitration in the enzyme as shown by Western blot analyses. LC/MS/MS analyses revealed that ONOO- treatment to aconitase resulted in nitration of tyrosines 151 and 472 and oxidation to sulfonic acid of cysteines 126 and 385. The latter is one of the three cysteine residues in aconitase that binds to the Fe-S cluster. All other modified tyrosine and cysteine residues were adjacent to the binding site, thus suggesting that these modifications caused conformational changes leading to active-site disruption. Aconitase cysteine thiol modifications other than oxidation to sulfonic acid, such as S-glutathionylation, also decreased aconitase activity, thus indicating that glutathionylation may be an important means of modulating aconitase activity under oxidative and nitrative stress. Taken together, these results demonstrate that the Fe-S cluster in the active site, cysteine 385 bound to the Fe-S cluster, and tyrosine and cysteine residues in the vicinity of the active site are important targets of oxidative and/or nitrative attack, which is selectively controlled by the mitochondrial matrix citrate levels. The mechanisms inherent in aconitase inactivation by ONOO- are discussed in terms of the mitochondrial matrix metabolic and thiol redox state.

Mechanisms of Liver Injury. III. Role of glutathione redox status in liver injury

“…Although sulfenic acid formation is readily reversible by GSH, it is unstable and can undergo further oxidation to sulfinic and sulfonic acids, which represent irreversible modifications. Recently, it has been shown that treatment of the mitochondrial protein aconitase with ONOO Ϫ results in sulfonic acid formation on cysteines residues and an inhibition of activity (9). ROS or RNS can also cause vicinal thiols or thiols from different proteins to undergo intra-or intermolecular disulfide bridge formation, respectively.…”

Nitro-fatty Acid Reaction with Glutathione and Cysteine

“…Gene expression of pro-inflammatory cytokines is also inhibited by electrophiles. For example, electrophilic alkylation of the pro-inflammatory transcription factor, nuclear factor-B (NF-B) modifies critical thiols in the p65 and p50 subunits, in turn suppressing DNA binding and NF-〉 target gene activation (12)(13)(14)(15)(16). Fatty acid-derived electrophiles also serve as ligands for receptors activated by the modulation of a critical Cys; for * This work was supported in part by National Institutes of Health Grants example, 15-deoxy-⌬ 12,14 -PGJ 2 (15dPGJ 2 ) binds to peroxisome proliferator-activated receptor-␥ (PPAR␥), increasing transcription of PPAR response element-regulated genes (17)(18)(19).…”

Chemoreception in the context of the general biology of ROS

“…Aside from the biological consequences that we will discuss below, inactivation of either form of aconitase can help to trace the origin, mitochondrial or cytoplasmic, of O 2 •− (Han et al, 2005) in any given situation (although escape of part of mitochondrially generated O 2 •− to the cytoplasm can blur the situation). For example, Kumar et al (2006) have observed that intermittent hypoxia caused a preferential decrease in the activity of mitochondrial aconitase suggesting that the main locus of O 2 •− generation in these situations is mitochondria.…”

“…Obviously, inactivation of mitochondrial aconitase leads to a decrease in the Krebs activity with the subsequent impairment of energy production. However, the extension of damage depends on several factors (Han et al, 2005): (a) activity of mitochondrial SOD, as for example null mice for mitochondrial SOD exhibited marked reduction (in comparison to controls) in the activity of aconitase as well in the iron-sulfur containing respiratory complexes I and II-III; (b) abundance of iron-sulfur enzymes (varies from tissue to tissue); and (c) the concentration of citrate which is protective and the presence of NO • . NO • can react with O 2 •− to form peroxynitrite (at a nearly diffusion controlled rate; ONOO − ; see below) which is very strong oxidant and nitrating agent capable of attacking the iron-sulfur clusters, nitrating tyrosine residues and oxidizing cysteine residues, and indeed producing loss of aconitase activity and other iron-sulfur clustercontaining enzymes (Han et al, 2005).…”

“…However, the extension of damage depends on several factors (Han et al, 2005): (a) activity of mitochondrial SOD, as for example null mice for mitochondrial SOD exhibited marked reduction (in comparison to controls) in the activity of aconitase as well in the iron-sulfur containing respiratory complexes I and II-III; (b) abundance of iron-sulfur enzymes (varies from tissue to tissue); and (c) the concentration of citrate which is protective and the presence of NO • . NO • can react with O 2 •− to form peroxynitrite (at a nearly diffusion controlled rate; ONOO − ; see below) which is very strong oxidant and nitrating agent capable of attacking the iron-sulfur clusters, nitrating tyrosine residues and oxidizing cysteine residues, and indeed producing loss of aconitase activity and other iron-sulfur clustercontaining enzymes (Han et al, 2005). It should be recalled that mitochondrial nitric oxide synthase generates NO • and therefore the formation of ONOO − is favored; in fact it has been estimated that 15% of the O 2 •− generated by mitochondria ends up as ONOO − and the reaming 85% as H 2 O 2 , generated by mitochondrial SOD, oxidation of iron-sulfur clusters and probably by reactions with other antioxidant molecules located in the mitochondrial interior.…”