Redox-Dependent Modulation of Aconitase Activity in Intact Mitochondria

Abstract

It has previously been reported that exposure of purified mitochondrial or cytoplasmic aconitase to superoxide (O(2)(-)(*) or hydrogen peroxide (H(2)O(2)) leads to release of the Fe-alpha from the enzyme's [4Fe-4S](2+) cluster and to inactivation. Nevertheless, little is known regarding the response of aconitase to pro-oxidants within intact mitochondria. In the present study, we provide evidence that aconitase is rapidly inactivated and subsequently reactivated when isolated cardiac mitochondria are treated with H(2)O(2). Reactivation of the enzyme is dependent on the presence of the enzyme's substrate, citrate. EPR spectroscopic analysis indicates that enzyme inactivation precedes release of the labile Fe-alpha from the enzyme's [4Fe-4S](2+) cluster. In addition, as judged by isoelectric focusing gel electrophoresis, the relative level of Fe-alpha release and cluster disassembly does not reflect the magnitude of enzyme inactivation. These observations suggest that some form of posttranslational modification of aconitase other than release of iron is responsible for enzyme inactivation. In support of this conclusion, H(2)O(2) does not exert its inhibitory effects by acting directly on the enzyme, rather inactivation appears to result from interaction(s) between aconitase and a mitochondrial membrane component responsive to H(2)O(2). Nevertheless, prolonged exposure of mitochondria to steady-state levels of H(2)O(2) or O(2)(-)(*) results in disassembly of the [4Fe-4S](2+) cluster, carbonylation, and protein degradation. Thus, depending on the pro-oxidant species, the level and duration of the oxidative stress, and the metabolic state of the mitochondria, aconitase may undergo reversible modulation in activity or progress to [4Fe-4S](2+) cluster disassembly and proteolytic degradation.

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

“…3B, the relative loss in enzyme activity during the first 5.0 min of reperfusion did not correspond with a similar increase in the level of the [3Fe-4S] 1ϩ form of aconitase. As a comparison, we have previously reported that, in mitochondria (30 mg of protein per ml required for EPR analysis) treated for 40 s with 2.0 mM H 2 O 2 in the presence of 2.0 mM citrate, Ϸ15% of aconitase is converted to the [3Fe-4S] 1ϩ cluster form of the enzyme (15). Ischemia͞reperfusion failed to evoke a signal of comparable magnitude (Fig.…”

“…These events are likely terminal in the dynamic response of aconitase to alterations in redox status. We have shown in vitro that aconitase can undergo reversible oxidative inhibition in the absence of alterations in the enzyme's [4Fe-4S] 2ϩ cluster (15). Reversible inhibition appears dependent on the interaction of aconitase with the mitochondrial iron-binding protein frataxin.…”

“…In vitro evidence indicates that frataxin can act as a chaperone protein, protecting aconitase from prooxidant-induced disassembly of the [4Fe-4S] 2ϩ cluster, and that frataxin is required for reversible modulation of enzyme activity (24). Nevertheless, depending on the magnitude and duration of the oxidative stress, reversible inhibition can progress to irreversible inactivation through [4Fe-4S] 2ϩ cluster disassembly, carbonylation, and ATP-dependent degradation (15). In the in vivo scenario of ischemia͞reperfusion, there are likely numerous variables, such , and R60, respectively).…”

“…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).…”

“…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). If the oxidative stress is prolonged or of sufficient magnitude, reversible modulation of aconitase activity progresses to irreversible inactivation and ATP-dependent degradation (15).…”