In vitro activation of apo-aconitase using a [4Fe-4S] cluster-loaded form of the IscU [Fe-S] cluster scaffolding protein

Biochemistry. 2007 Jun 12;46(23):6812-21. doi: 10.1021/bi6026665. Epub 2007 May 17.

Abstract

Genetic experiments have established that IscU is involved in maturation of [Fe-S] proteins that require either [2Fe-2S] or [4Fe-4S] clusters for their biological activities. Biochemical studies have also shown that one [2Fe-2S] cluster can be assembled in vitro within each subunit of the IscU homodimer and that these clusters can be reductively coupled to form a single [4Fe-4S] cluster. In the present work, it is shown that the [4Fe-4S] cluster-loaded form of A. vinelandii IscU, but not the [2Fe-2S] cluster-loaded form, can be used for intact cluster transfer to an apo form of A. vinelandii aconitase A, a member of the monomeric dehydratase family of proteins that requires a [4Fe-4S] cluster for enzymatic activity. The rate of [4Fe-4S] cluster transfer from IscU to apo-aconitase A was not affected by the presence of the HscA/HscB co-chaperone system and MgATP. However, an altered form of a [4Fe-4S] cluster-containing IscU, having the highly conserved aspartate-39 residue substituted with alanine, is an effective inhibitor of wild-type [4Fe-4S] cluster-loaded IscU-directed activation of apo-aconitase A. In contrast, neither the clusterless form of IscU nor the [2Fe-2S] cluster-loaded form of IscU is an effective inhibitor of IscU-directed apo-aconitase A activation. These results are interpreted to indicate that the [2Fe-2S] and [4Fe-4S] cluster-loaded forms of IscU adopt different conformations that provide specificity with respect to the maturation of [2Fe-2S] and [4Fe-4S] centers in proteins.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Aconitate Hydratase / metabolism*
  • Apoproteins / chemistry
  • Apoproteins / isolation & purification
  • Apoproteins / metabolism*
  • Azotobacter vinelandii / metabolism
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Iron-Sulfur Proteins / chemistry
  • Iron-Sulfur Proteins / isolation & purification
  • Iron-Sulfur Proteins / metabolism*
  • Kinetics
  • Nuclear Matrix-Associated Proteins / chemistry
  • Nuclear Matrix-Associated Proteins / isolation & purification
  • Nuclear Matrix-Associated Proteins / metabolism*
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Spectrophotometry
  • Thermodynamics

Substances

  • Apoproteins
  • Bacterial Proteins
  • Iron-Sulfur Proteins
  • Nuclear Matrix-Associated Proteins
  • Protein Subunits
  • Recombinant Proteins
  • Aconitate Hydratase