In vitro activation of apo-aconitase using a [4Fe-4S] cluster-loaded form of the IscU [Fe-S] cluster scaffolding protein.
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.
Unciuleac
Mihaela-Carmen
MC
Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061, USA.
Chandramouli
Kala
K
Naik
Sunil
S
Mayer
Suzanne
S
Huynh
Boi Hanh
BH
Johnson
Michael K
MK
Dean
Dennis R
DR
D000154:Aconitate Hydratase / Q000378:metabolism*; D001059:Apoproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016948:Azotobacter vinelandii / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D034521:Nuclear Matrix-Associated Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013816:Thermodynamics
D001059:Apoproteins; D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; D034521:Nuclear Matrix-Associated Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D000154:Aconitate Hydratase