Molecular forms of aconitase and their interconversions

Area of Science:

• Biochemistry
• Enzymology
• Mitochondrial Function

Background:

• Mammalian aconitase, isolated via traditional methods, exhibits low activity and possesses an oxidized [3Fe-4S]+ cluster.
• Understanding the activation mechanism of aconitase is crucial for comprehending its role in cellular metabolism and iron-sulfur cluster biology.

Purpose of the Study:

• To investigate the activation process of inactive mammalian aconitase.
• To elucidate the role of iron (Fe2+) and dithionite in enzyme activation and conformational changes.
• To characterize the structural changes of the iron-sulfur cluster during activation.

Main Methods:

• Enzyme activity assays.
• Tryptophan fluorescence spectroscopy.
• Electron paramagnetic resonance (EPR) spectroscopy of the iron-sulfur cluster.
• Incubation with dithionite and varying concentrations of Fe2+.

Main Results:

• Full aconitase activity is restored with one electron per [3Fe-4S] cluster and at least 0.6 Fe2+ per molecule, forming [4Fe-4S]2+ clusters.
• Reduction alone (without Fe2+) yields up to 70% activity but requires more reductant, suggesting internal iron redistribution.
• Conformational changes (indicated by fluorescence) occur concurrently with activation when Fe2+ is present, but precede activation in its absence, as shown by iron chelator experiments.

Conclusions:

• Aconitase activation involves the formation of [4Fe-4S] clusters, requiring both reduction and iron insertion.
• The conformational change of the aconitase protein is triggered by Fe-S cluster reduction and precedes iron incorporation.
• Iron chelators inhibit activation but not the initial conformational change, supporting a model where protein rearrangement occurs before Fe2+ insertion.