The inactive form of aconitase contains a [3Fe-4S] cluster held in place by cysteines 358, 421, and 424. When the enzyme is activated, a fourth Fe is inserted into the cluster to form a cube-like structure. The fourth Fe is also attached to a hydroxyl group as its fourth ligand. Upon binding of the substrate (shown in Figure 1), the Fe changes from a hydroxyl-bound, four-coordinate Fe state to a water-bound, six-coordinate Fe state. The proton shifts in this change are coordinated by the histidine-carboxylate pairs which can either donate or accept protons.⁷

The reaction to form cis-aconitate to form isocitrate (shown in Figure 2) is initiated when serine 642 abstracts the proton from the beta carbon of the isocitrate. This transfer causes the formation of an aci-acid intermediate which collapses with the cleavage of the alpha carbon-OH bond to form cis-aconitate. To form citrate, the fourth domain of aconitase opens up via its linker peptide and allows the cis-aconitate to be displaced and replaced by another cis-aconitate that is flipped over 180 degrees about the alpha carbon-beta carbon bond. A reversal of the steps used to form the cis-aconitate from isocitrate yields the product citrate. The whole reaction can be done in reverse to form isocitrate from citrate.⁷

Nitrocitrate, nitroisocitrate, trans-aconitate, and (-) erythro- 2-fluorocitrate are tight-binding inhibitors of aconitase. Nitrocitrate, nitroisocitrate, and trans-aconitate have a trigonal carbon adjacent to the carbon with the hydroxyl group and are considered to be transition state analogs of the carbanion intermediate formed by citrate or isocitrate during the reaction. The reactions of nitrocitrate and nitroisocitrate inhibitors with aconitase are often studied to learn more about the transition state mechanism of the enzyme.⁶

Fluoroacetate is an extremely poisonous compound that blocks the citric acid cycle¹ causing a buildup of citrate that can lead to convulsive seizures.⁹ Fluoroacetate has been called a "Trojan horse inhibitor." It enters the citric acid cycle and is converted by acetyl-CoA-synthetase to fluoroacetyl-CoA which is converted by citrate synthase to fluorocitrate. It isn't inhibitory until it reacts with aconitase and blocks the citric acid cycle.¹ Fluorocitrate is converted to fluoro-cis-aconitate and then to 4-hydroxy-trans-aconitate (HTn) which binds tightly to aconitase and inhibits its action. It does this by displacing the double bond closest to the Fe and preventing the hydration of the double bond.⁹

Examples of the inhibitors trans-aconitate, 4-hydroxy-aconitate, and nitroisocitric acid bound to the Fe-S cluster can be seen in the models to the right.