456 IRON-CONTAINING PROTEINS AND ENZYMES
(S =^1 / 2 ), [Fe 4 S 4 ] 2+ ( S = 0), and [Fe 4 S 4 ] + ( S =^1 / 2 ), containing iron ions in +2, +3,
and +2.5 states.
The enzyme aconitase contains four domains: Domains 1, 2, and 3 are
closely associated with the [4Fe – 4S] iron – sulfur cluster that resides in the
active site, while domain 4 is attached by a peptide linker to create a large
cleft that leads toward the active site. In 1992, the C. D. Stout group solved the
X - ray crystallographic structure of porcine heart mitochondrial aconitase with
bound substrate isocitrate (PDB: 7ACN).^172 The 82.8 - kDa protein contains 754
amino acid residues in addition to an isocitrate group and an [Fe 4 S 4 ] 2+ iron –
sulfur cluster. The same research group solved the structure of bovine heart
enzyme in complex with the reaction intermediate analog and inhibitor nitro-
isocitrate bound (PDB: 8ACN). Both substrate compounds bind to the unique
iron atom in a [4Fe – 4S] iron – sulfur cluster — the Fe not coordinated by a cys-
teine ligand — via hydroxyl and carbonyl oxygens. The substrate - coordinating
Fe a atom coordinates an α - carboxyl oxygen of isocitrate, a β - carboxyl oxygen
of citrate, and either the α - or β - carboxyl oxygen of cis - aconitate as well as a
substrate hydroxyl and H 2 O from solvent. Twenty - three amino acid residues
from all four aconitase domains contribute interactions within the [4Fe – 4S]
cluster - containing active site.
Figure 7.50 shows some of the important amino acid residues surrounding
the isocitrate substrate (represented in ball - and - stick format with black carbon
atoms). The iron – sulfur cluster, also in ball - and - stick format, shows sulfur
atoms colored yellow and shows iron atoms in orange - brown. Residues cys358,
cys421, cys424, asn258, and asn446 contribute ligation and other interactions
with the iron – sulfur cluster — carbon atoms colored orange in Figure 7.50.
Residues arg447, arg452, arg580, arg644, gln72, ser166, and ser643 participate
in substrate recognition — carbon atoms colored green in Figure 7.50. Residues
ala74, asp568, ser571, and thr567 contribute hydrogen bonds to active site
residue side chains. Ser642 and three acidic side - chain histidine pairs (asp100 –
his101, asp165 – his147 and glu262 – his167) contribute specifi cally to the cata-
lytic reaction shown in equation 7.13 and Figure 7.49. (The carbon atoms of
these residues are colored magenta in Figure 7.50. For clarity, not every aa
residue is shown.) For example, ser642 appears to be stabilized as an alkoxide
to assist in proton abstraction from C β of the isocitrate moiety. In addition to
the catalytic group abstracting the proton, another catalytic group must pro-
tonate the substrate hydroxyl to remove this group as a water molecule. An
extensive hydrogen - bonding network is involved in the protonation of sub-
strate hydroxyl and Fe - bound hydroxyls. Some typical PDB: 7ACN bond
lengths are displayed in Table 7.11.
The X - ray structures of other aconitases have appeared in the literature.
Recently, the crystal structure of human iron regulatory protein, IRP1, in its
aconitase form, has been published.^173 Iron regulatory proteins (IRPs) control
the translation of proteins involved in iron uptake, storage, and utilization by
binding to specifi c noncoding sequences of the corresponding mRNAs known