A Tour of Aconitase

Please be patient while the structures in the left frame load. In order to display all the structures in the tour properly, press 'View' buttons below in order (from 1 to the end).

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:

There is a polypeptide segment of 25 amino acids (blue segment) that links the C-terminal domain (in green) to domains 1-3 (all three in white).

The active site of aconitase is a [4Fe-4S] cluster located in the N-terminal domain.

The inactive form of aconitase contains a [3Fe-4S] cluster. Its structure is similar to that of a cubane [4Fe-4S] cluster except that it is missing one Fe atom. It is ligated with three cysteine residues (Cys 358, 421, 424) of domain 3. This form of aconitase is important for the regulation of iron levels in the living organisms.

Domain 3 provides cysteines which anchor the cluster to the protein while domains 1, 2 and 4 provide a number of other amino acid residues around the cluster. The function of some of them will be discussed further below. At the moment just note the large number of nitrogen (blue) and oxygen (red) atoms oriented towards the active center. The presence of the polar groups from these residues makes the cavity around the Fe-S cluster highly hydrophilic. Also note that there are no additional cysteine residues which could serve as additional ligands inside the cavity.

The enzyme is activated once the [3Fe-4S] cluster of the inactive form is converted to the [4Fe-4S] cluster.

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.

The -OH group acts as a hydrogen bond donor to Asp 165. Because Fea 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.

  The enzyme substrate, citrate, bonds to Fea via a Cβ carboxyl oxygen and Cβ hydroxyl group. A hydroxyl group is still coordinated making the geometry around Fea octahedral.

  One of the steps in the citrate-to-isocitrate conversion mechanism is the abstraction of a proton from the Cα carbon atom. Structural and spectroscopic investigations suggest that the base accepting the proton is the alkoxide form of serine 642. The display on the left shows the structure of a mutant protein in which Ser642 has been replaced by alanine (Ser642Ala or S642A mutation). The mutation reduces the aconitase reactivity by 5 orders of magnitude.

 Simultaneously or following the H+ abstraction, the hydroxyl group on Cβ is protonated and eliminated as water giving the intermediate cis-aconitate. [1]. 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.

 This is the structure of aconitase active site with isocitrate bound. This time the substrate bonds to the metal via the Cα-carboxyl oxygen and the Cα-hydroxyl group.

This is a wireframe display of activated aconitase for your further study.

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 structure of inactive and active forms of aconitase: Robbins, A.H.; Stout, C.D. (1989) Proc. Natl. Acad. Sci. USA, 86, 3639-3634 (PDB ids: 5ACN - inactive form, 6ACN - active form) and see also Robbins, A.H.; Stout, C.D. (1989) Proteins, 5, 289-312.

The crystal structure of aconitase with isocitrate bound: Lauble, H. et al. (1992) Biochemistry 31(10), 2735 - 2748 (PDB id 7ACN).

The crystal structure of aconitase with citrate bound: Lloyd, S.J. et al. (1999) Protein Science 8, 2665-2662 (PDB id 1C96).

Cytoplasmatic aconitase as iron regulatory protein (IRP)

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.

Aconitase - A Model Complex

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.

  The proposed conversion was verified when Holm's group prepared [Fe3S4(SR)4]3- (R = Ph, Et) complexes [2]. 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.

The synthesis of model complex analogues for both the active and inactive forms of aconitase proved to be a challenging task. The main synthetic problem was to obtain a 4Fe - 4S core in which three iron ions would have identical environments while the fourth one would have a different terminal ligation (so-called 3 : 1 site-differentiated [4Fe - 4S] clusters). The solution came again from Holm's group with the synthesis of the semi-rigid trithiol ligand (L-S3). [3]   The synthetic procedure, starting from 1,3,5-trifluorobenzene and 1,3-dimethylbenzene, has 12 steps.

Treatment of the DMF solution of trithiol ligand with (Ph4P)2[Fe4S4(SEt)4] 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-.[4]

Treatment of [Fe4S4(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. [5]

[1] The structure of cis-aconitate complex is presently not available. However, the structure of trans-aconitate is. See: Lauble, H. et al. (1994) J. Mol. Bio. 237, 437 (PDB id 1ACO).

[2] Hagen, K.S. et al. (1983) J. Am. Chem. Soc. 105(12) 3905 - 3913

[3] Stack, T.D.P. et al. (1988) J. Am. Chem. Soc. 110(8) 2484 - 2494

[4] Zhou, J. et al. (1995) J. Am. Chem. Soc. 117(45) 11353 - 11354

[5] Zhou, J. et al. (1996) J. Am. Chem. Soc. 118(8) 1966 - 1980

Copyright Robert H. Morris and Alen Hadzovic, 1998, 2009, 2011, 2017.