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Analysis of the Iron‐Sulfur Cluster in Human Cytoplasmic Aconitase by Electron Paramagnetic Resonance Spectroscopy

Human recombinant cytoplasmic aconitase, also known as human iron regulatory protein 1 (hIRP‐1), was studied in vitro by electron paramagnetic resonance (EPR) spectroscopy to determine the effects of urea on iron‐sulfur cluster assembly. The bifunctional protein functions as either an enzyme that catalyzes the isomerization of citrate in the cytoplasm or as an iron‐responsive element‐binding protein in human iron metabolism and regulation. Protein behavior is regulated by the assembly and disassembly of the [4Fe‐4S]2+ cluster, which is retained and active at high cellular iron concentrations. At low iron levels, the cluster is disassembled and the protein loses its enzymatic function. Although the [4Fe‐4S]2+ is diamagnetic, alternate forms of the cluster are paramagnetic and may be observed by EPR as the protein is denatured. EPR spectra obtained at high and low fields were used to characterize the iron‐sulfur cluster. Titration with urea reveals the existence of a paramagnetic species that appears at about 2.0 M urea and disappears above 2.5 M urea, consistent with the gradual disassembly of the Fe‐S cluster. The changes in the Fe‐S cluster reported by EPR correspond closely to protein conformational changes detected by intrinsic tryptophan fluorescence and active‐site structure monitored by enzyme activity. Experiments are underway to determine the effects of substrate and inhibitor on stability of the cluster in the presence of urea.