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The aconitase family of proteins catalyzes the interconversion of citrate and isocitrate via cis-aconitate intermediate in the second and the third steps of the Krebs' cycle. Aconitases can be mitochondrial or cytoplasmic. The cytoplasmic aconitases are bifunctional: they catalyze the citrate/isocitrate interconversion but also act as iron regulatory proteins (IRP) (see below).
Regardless of their occurrence in the cell, all aconitases are monomeric proteins containing approximately 750 residues and with a weight of 83 kDa. The display on the left shows the wireframe model of the inactive form of aconitase isolated from the mitochondria of pig's heart cells.
The structure of aconitase consists of 4 domains:
The new Fe(II) ion closes the cubane structure and is usually referred to as Fea or Fe4. When the fourth Fe ion is inserted into the empty corner no significant change of the cluster geometry takes place. Fea has a hydroxyl anion coordinated but has no amino acid ligands from the protein.
a lacks an amino acid ligand and is directed towards the active site cavity, its position and coordination environment are ideal for the interaction with a substrate.
a via a Cβ carboxyl oxygen and Cβ hydroxyl group. A hydroxyl group is still coordinated making the geometry around Fea octahedral.
+ abstraction, the hydroxyl group on Cβ is protonated and eliminated as water giving the intermediate cis-aconitate. . Some of the residues assisting in the protonation and elimination process are shown in the left frame. Note the presence of nitrogen (blue) and oxygen (red) atoms around the coordinated citrate! The oxygen atom labeled "- H2O" belongs to the hydroxyl group that is going to be eliminated as a water molecule.
If you would like to find out more about aconitase, its structure and function see Beinert, H. et al. (1996) Chem. Rev. 96, 2335 - 2373.
The control of intracellular iron levels depends on the function of two proteins: the transferrin receptor, which recognizes the iron-loaded transferrin and moves it into the cells, and the protein responsible for iron storage - ferritin. The concentration of these proteins in the cell is regulated by IRPs. These proteins are cytoplasmatic aconitases that lose the Fe-S cluster when the iron level in the cell is low. IRP is inactivated when the available iron levels for complexation is plentiful, giving rise to the reconstruction of the [4Fe-4S] cluster and activation of aconitase.
The mRNAs for transferrin receptor and for ferritin contain so-called iron-responsive elements (IREs). These IREs bind with high affinity to the IRP. At low iron levels, the IRP binds to the IRE in the 5“ region of ferritin mRNA, resulting in the blockage of protein translation. Active IRP also binds to the IREs in the 3“ region of tranferrin receptor mRNA, resulting in the protection of the mRNA from being degraded by nucleases. Thus, the interplay of the [4Fe-4S] cluster assembly and disassembly pathways regulates the levels of transferrin receptor and ferritin in a complementary sense, supporting either the uptake or storage of iron.
During the early days of research on aconitase it was noted that the inactive enzyme changed colour from brown to purple at pH > 9. The spectroscopic studies (EPR, Raman, Mössbauer) suggested that the reason behind this drastic change in the protein colour is the conversion of the [3Fe - 4S] cluster geometry from cuboidal to linear. However, in the absence of crystallographic data on the purple form of the aconitase, this idea could not be confirmed.
3S4(SR)4]3- (R = Ph, Et) complexes . The structure of complex with R = Ph is shown here.
The complex contains a [Fe(µ2-S)2Fe(µ2-S)2Fe]+ cluster. The terminal two Fe ions are coordinated by two thiolate ligands. Each Fe atom has a distorted tetrahedral geometry. The remarkable similarities in spectroscopic properties between this model compound and the purple form of aconitase not only confirmed the hypothesis but also implied that the linear [3Fe - 4S ] cluster has 4 cysteine ligands (compared to three for the cuboidal form). The later study established that the linear cluster retains two of the cysteine residues from original structure (Cys421 and Cys424) while the other two (Cys250 and Cys257) come from domain 2. A considerable protein conformational change is required to bring these new ligands into position.
3).  The synthetic procedure, starting from 1,3,5-trifluorobenzene and 1,3-dimethylbenzene, has 12 steps.
followed by addition of pivaloyl chloride afforded the first synthetic model of the aconitase active form: [Fe4S4(L-S3)Cl]2-. The complex contains the necessary 3 : 1 site-differentiated [4Fe - 4S] cluster with one unique Fe site terminally ligated to Cl-. It has been shown that chloride can be easily exchanged with other ligands, for example with EtS-.
4S4(L-S3)(SEt)]2- with (Et3NH)(OTf) (OTf = triflate, CF3SO3-) affords [Fe4S4(L-S3)(OTf)]2-. Treatment of the triflate complex with 2-3 equivalents of (Et4N)(Meida) (Meida = N-methylimidodiacetate, a good Fe2+ chelator) readily abstracts the unique Fe(II) ion from the [4Fe - 4S] cluster converting the starting compound into a model complex for inactive aconitase containing a cuboidal [3Fe - 4S] cluster.