Tags

Type your tag names separated by a space and hit enter

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.
J Biol Chem. 2002 Mar 01; 277(9):7246-54.JB

Abstract

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.

Authors+Show Affiliations

Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612, USA.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

11744706

Citation

Brown, Nina M., et al. "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." The Journal of Biological Chemistry, vol. 277, no. 9, 2002, pp. 7246-54.
Brown NM, Kennedy MC, Antholine WE, et al. 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. J Biol Chem. 2002;277(9):7246-54.
Brown, N. M., Kennedy, M. C., Antholine, W. E., Eisenstein, R. S., & Walden, W. E. (2002). 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. The Journal of Biological Chemistry, 277(9), 7246-54.
Brown NM, et al. 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. J Biol Chem. 2002 Mar 1;277(9):7246-54. PubMed PMID: 11744706.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Brown,Nina M, AU - Kennedy,M Claire, AU - Antholine,William E, AU - Eisenstein,Richard S, AU - Walden,William E, Y1 - 2001/12/13/ PY - 2001/12/18/pubmed PY - 2002/4/2/medline PY - 2001/12/18/entrez SP - 7246 EP - 54 JF - The Journal of biological chemistry JO - J Biol Chem VL - 277 IS - 9 N2 - 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. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/11744706/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_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(19)82280-9 DB - PRIME DP - Unbound Medicine ER -