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Originally published In Press as doi:10.1074/jbc.M110282200 on December 13, 2001

J. Biol. Chem., Vol. 277, Issue 9, 7246-7254, March 1, 2002
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Detection of a [3Fe-4S] Cluster Intermediate of Cytosolic Aconitase in Yeast Expressing Iron Regulatory Protein 1
INSIGHTS INTO THE MECHANISM OF Fe-S CLUSTER CYCLING*

Nina M. BrownDagger §, M. Claire Kennedy||, William E. Antholine**, Richard S. EisensteinDagger Dagger , and William E. WaldenDagger §§

From the Dagger  Department of Microbiology and Immunology, University of Illinois, Chicago, Illinois 60612, the  Biochemistry Department, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, the ** Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and the Dagger Dagger  Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706

Interconversion of iron regulatory protein 1 (IRP1) with cytosolic aconitase (c-aconitase) occurs via assembly/disassembly of a [4Fe-4S] cluster. Recent evidence implicates oxidants in cluster disassembly. We investigated H2O2-initiated Fe-S cluster disassembly in c-aconitase expressed in Saccharomyces cerevisiae. A signal for [3Fe-4S] c-aconitase was detected by whole-cell EPR of aerobically grown, aco1 yeast expressing wild-type IRP1 or a S138A-IRP1 mutant (IRP1S138A), providing the first direct evidence of a 3Fe intermediate in vivo. Exposure of yeast to H2O2 increased this 3Fe c-aconitase signal up to 5-fold, coincident with inhibition of c-aconitase activity. Untreated yeast expressing IRP1S138D or IRP1S138E, which mimic phosphorylated IRP1, failed to give a 3Fe signal. H2O2 produced a weak 3Fe signal in yeast expressing IRP1S138D. Yeast expressing IRP1S138D or IRP1S138E were the most sensitive to inhibition of aconitase-dependent growth by H2O2 and were more responsive to changes in media iron status. Ferricyanide oxidation of anaerobically reconstituted c-aconitase yielded a strong 3Fe EPR signal with wild-type and S138A c-aconitases. Only a weak 3Fe signal was obtained with S138D c-aconitase, and no signal was obtained with S138E c-aconitase. This, paired with loss of c-aconitase activity, strongly argues that the Fe-S clusters of these phosphomimetic c-aconitase mutants undergo more complete disassembly upon oxidation. Our results demonstrate that 3Fe c-aconitase is an intermediate in H2O2-initiated Fe-S cluster disassembly in vivo and suggest that cluster assembly/disassembly in IRP1 is a dynamic process in aerobically growing yeast. Further, our results support the view that phosphorylation of IRP1 can modulate its response to iron through effects on Fe-S cluster stability and turnover.

* This work was supported by National Institutes of Health Grants DK47281 (to W. E. W.), DK47219 (to R. S. E.), and GM51831 (to M. C. K.). The facilities of the National Biomedical ESR Center at the Medical College of Wisconsin were supported by National Institutes of Health Grant RR01008.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: Dept. of Chemistry, Northwestern University, 2145 Sheridan Rd., Evanston, IL 60208.

|| Present address: Dept. of Chemistry, Gannon University, Erie, PA 16541.

§§ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612. Tel.: 312-996-8576; Fax: 312-996-6415; E-mail: wwalden@uic.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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