Aconitase assay

Gardner and Fridovich [85] proposed that the inactivation of aconitase might be used as an assay of superoxide formation in cells. The mechanism of the interaction of superoxide with aconitases has been considered in Chapter 21. As follows from data presented in that chapter, peroxynitrite is also able to inactivate aconitases rapidly therefore, this method cannot be a specific assay of superoxide detection. 

Figure 4. Oxidative titration of activated aconitase with potassium fenicyanide. Aliquots of activated aconitase and a measured amount of K3Fe(CN)6 were added to individual EPR tubes, assayed for enzymatic activity ( ), and frozen. After deteimination of the number of spins by EPR at g = 2.01 (x), the samples were thawed and assayed again (o). (Reproduced with permission from Ref. 47. Copyright 1983 American Society Biological Chemists.)...

By this time it was demonstrated that the [3Fe-4S]W+ form of aconitase is inactive, while the [4Fe-4S]2+ form is active. How is the activity of the enzyme affected by the oxidation state of the [4Fe-4S] cluster Because the active enzyme contains a [4Fe-4S]2+ cluster, either the 3+ or 1+ oxidation states may also be stable. The 3+ state is unstable since oxidation of the [4Fe-4S]2+ resulted in the immediate loss of a ferrous ion and conversion to a [3Fe-4S]i+ cluster (46,47). However, reduction of active aconitase by sodium dithionite or photoreduction in the presence of deazaflavin produced in high yields an EPR signal characteristic for [4Fe-4S]l+ clusters (47). When active enzyme within an anaerobic assay cuvette was photoreduced, the activity of the enzyme dropped to 1/3 of its initial value. Further photoreduction resulted in cluster destruction. Then, if the enzyme is reoxidized with air, the activity returned to its original value. This demonstrated that the redox state of the cluster can modulate the enzyme activity. A scheme summarizing the cluster interconversions and various redox states of the Fe-S cluster of aconitase is shown below. 

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