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The SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster transfer.

ABSTRACT: Iron-sulfur clusters are key iron cofactors in biological pathways ranging from nitrogen fixation to respiration. Because of the toxicity of ferrous iron and sulfide to the cell, in vivo Fe-S cluster assembly transpires via multiprotein biosynthetic pathways. Fe-S cluster assembly proteins traffic iron and sulfide, assemble nascent Fe-S clusters, and correctly transfer Fe-S clusters to the appropriate target metalloproteins in vivo. The Gram-negative bacterium Escherichia coli contains a stress-responsive Fe-S cluster assembly system, the SufABCDSE pathway, that functions under iron starvation and oxidative stress conditions that compromise Fe-S homeostasis. Using a combination of protein-protein interaction and in vitro Fe-S cluster assembly assays, we have characterized the relative roles of the SufBCD complex and the SufA protein during Suf Fe-S cluster biosynthesis. These studies reveal that SufA interacts with SufBCD to accept Fe-S clusters formed de novo on the SufBCD complex. Our results represent the first biochemical evidence that the SufBCD complex within the Suf pathway functions as a novel Fe-S scaffold system to assemble nascent clusters and transfer them to the SufA Fe-S shuttle.

SUBMITTER: Chahal HK

PROVIDER: S-EPMC2783425 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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The SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster transfer.

Chahal Harsimranjit K HK Dai Yuyuan Y Saini Avneesh A Ayala-Castro Carla C Outten F Wayne FW

Biochemistry 20091101 44

Iron-sulfur clusters are key iron cofactors in biological pathways ranging from nitrogen fixation to respiration. Because of the toxicity of ferrous iron and sulfide to the cell, in vivo Fe-S cluster assembly transpires via multiprotein biosynthetic pathways. Fe-S cluster assembly proteins traffic iron and sulfide, assemble nascent Fe-S clusters, and correctly transfer Fe-S clusters to the appropriate target metalloproteins in vivo. The Gram-negative bacterium Escherichia coli contains a stress- ...[more]

PMID: 19810706

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Project description:Mitochondrial Fe-S cluster biosynthesis is accomplished within yeast utilizing the biophysical attributes of the "Isu1" scaffold assembly protein. As a member of a highly homologous protein family, Isu1 has sequence conservation between orthologs and a conserved ability to assemble [2Fe-2S] clusters. Regardless of species, scaffold orthologs have been shown to exist in both "disordered" and "structured" conformations, a structural architecture that is directly related to conformations utilized during Fe-S cluster assembly. During assembly, the scaffold helps direct the delivery and utilization of Fe(ii) and persulfide substrates to produce [2Fe-2S] clusters, however Zn(ii) binding alters the activity of the scaffold while at the same time stabilizes the protein in its structured state. Additional studies confirm Zn binds to the scaffold's Cys rich active site, and has an impact on the protein's ability to make Fe-S clusters. Understanding the interplay between Fe(ii) and Zn(ii) binding to Isu1 in vitro may help clarify metal loading events that occur during Fe-S cluster assembly in vivo. Here we determine the metal : protein stoichiometry for Isu1 Zn and Fe binding to be 1 : 1 and 2 : 1, respectively. As expected, while Zn binding shifts the Isu1 to its structured state, folding is not influenced by Fe(ii) binding. X-ray absorption spectroscopy (XAS) confirms Zn(ii) binds to the scaffold's cysteine rich active site but Fe(ii) binds at a location distinct from the active site. XAS results show Isu1 binding initially of either Fe(ii) or Zn(ii) does not significantly perturb the metal site structure of alternate metal. XAS confirmed that four scaffold orthologs bind iron as high-spin Fe(ii) at a site composed of ca. 6 oxygen and nitrogen nearest neighbor ligands. Finally, in our report Zn binding dramatically reduces the Fe-S cluster assembly activity of Isu1 even in the presence of frataxin. Given the Fe-binding activity we report for Isu1 and its orthologs here, a possible mechanism involving Fe(ii) transport to the scaffold's active site during cluster assembly has been considered.

Dataset Information

The SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster transfer.

Publications

The SufBCD Fe-S scaffold complex interacts with SufA for Fe-S cluster transfer.

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