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.
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 H(2)O(2)-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 (IRP1(S138A)), providing the first direct evidence of a 3Fe intermediate in vivo. Exposure of yeast to H(2)O(2) increased this 3Fe c-aconitase signal up to 5-fold, coincident with inhibition of c-aconitase activity. Untreated yeast expressing IRP1(S138D) or IRP1(S138E), which mimic phosphorylated IRP1, failed to give a 3Fe signal. H(2)O(2) produced a weak 3Fe signal in yeast expressing IRP1(S138D). Yeast expressing IRP1(S138D) or IRP1(S138E) were the most sensitive to inhibition of aconitase-dependent growth by H(2)O(2) 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 H(2)O(2)-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.
Brown
Nina M
NM
Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612, USA.
Kennedy
M Claire
MC
Antholine
William E
WE
Eisenstein
Richard S
RS
Walden
William E
WE
D000154:Aconitate Hydratase / Q000378:metabolism*; D001224:Aspartic Acid / Q000737:chemistry; D002455:Cell Division; D003593:Cytoplasm / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D018698:Glutamic Acid / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000378:metabolism; D007501:Iron / Q000378:metabolism*; D035941:Iron Regulatory Protein 1; D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010766:Phosphorylation; D016601:RNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry*; D017382:Reactive Oxygen Species; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013997:Time Factors
D035925:Iron-Regulatory Proteins; D007506:Iron-Sulfur Proteins; D016601:RNA-Binding Proteins; D017382:Reactive Oxygen Species; D001224:Aspartic Acid; D018698:Glutamic Acid; D012694:Serine; D006861:Hydrogen Peroxide; D007501:Iron; D000154:Aconitate Hydratase; D035941:Iron Regulatory Protein 1; D010100:Oxygen