Numerous degenerative disorders are associated with elevated levels of prooxidants and declines in mitochondrial aconitase activity. Deficiency in the mitochondrial iron-binding protein frataxin results in diminished activity of various mitochondrial iron-sulfur proteins including aconitase. We found that aconitase can undergo reversible citrate-dependent modulation in activity in response to pro-oxidants. Frataxin interacted with aconitase in a citrate-dependent fashion, reduced the level of oxidant-induced inactivation, and converted inactive [3Fe-4S]1+ enzyme to the active [4Fe-4S]2+ form of the protein. Thus, frataxin is an iron chaperone protein that protects the aconitase [4Fe-4S]2+ cluster from disassembly and promotes enzyme reactivation.

Restoration of blood flow to ischemic myocardial tissue results in an increase in the production of oxygen radicals. Highly reactive, free radical species have the potential to damage cellular components. Clearly, maintenance of cellular viability is dependent, in part, on the removal of altered protein. The proteasome is a major intracellular proteolytic system which degrades oxidized and ubiquitinated forms of protein. Utilizing an in vivo rat model, we demonstrate that coronary occlusion/ reperfusion resulted in declines in chymotrypsin-like, peptidylglutamyl-peptide hydrolase, and trypsin-like activities of the proteasome as assayed in cytosolic extracts. Analysis of purified 20 S proteasome revealed that declines in peptidase activities were accompanied by oxidative modification of the protein. We provide conclusive evidence that, upon coronary occlusion/ reperfusion, the lipid peroxidation product 4-hydroxy-2-nonenal selectively modifies 20 S proteasome ␣-like subunits iota, C3, and an isoform of XAPC7. Occlusion/ reperfusion-induced declines in trypsin-like activity were largely preserved upon proteasome purification. In contrast, loss in chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities observed in cytosolic extracts were not evident upon purification. Thus, decreases in proteasome activity are likely due to both direct oxidative modification of the enzyme and inhibition of fluorogenic peptide hydrolysis by endogenous cytosolic inhibitory protein(s) and/or substrate(s). Along with inhibition of the proteasome, increases in cytosolic levels of oxidized and ubiquitinated protein(s) were observed. Taken together, our findings provide insight into potential mechanisms of coronary occlusion/reperfusion-induced proteasome inactivation and cellular consequences of these events.Restoration of coronary blood flow to previously ischemic cardiac tissue is often associated with declines in cardiac function, including myocardial stunning, ventricular arrhythmias, hemodynamic abnormalities, and, in the long term, development of heart failure (1, 2). This paradoxical phenomenon, broadly termed ischemia/reperfusion injury, is accompanied by dramatic increases in tissue levels of free radicals as well as byproducts of lipid peroxidation (1-9). Due to the high reactivity of these species (10 -12), it has been proposed that free radical events play a key role in myocardial ischemia/reperfusion injury. Nevertheless, mechanisms by which free radicals alter cardiac function have not been fully elucidated. Free radical modification of protein can alter and, in many cases, inhibit, protein activity (10 -15). In addition, oxidatively crosslinked protein(s) are resistant to proteolytic degradation (16 -20). Thus, the presence of oxidized protein could impact cellular function by changing the catalytic and/or regulatory properties of specific proteins and by placing abnormal demands on finite cellular volume. Clearly, the level of oxidatively modified protein reflects the balance between free radical damage and p...