Insights into the structure of the diiron site of RIC from Escherichia coli

Abstract

Edited by Miguel De la RosaKeywords: RIC Diiron Nitrosative and oxidative stress Iron repair Iron-sulphur a b s t r a c t Repair of Iron Centres (RICs) are a widely-spread family of diiron proteins involved in the protection of iron-sulphur-containing enzymes from nitrosative and oxidative stress. Here, homology-based modelling was used to predict putative ligands of the RIC diiron centre in E. coli. Site-directed mutagenesis studies showed that several conserved residues modulate the spectroscopic properties of the diiron centre, and mutations in H129, E133 and E208 abrogated RIC ability to protect aconitase. Taken together, these data led to a structural model of a diiron centre inserted in a four-helix bundle fold and coordinated by H84, H129, H160, H204, E133 and E208. Moreover, two l-carboxylate bridges involving E133 and E208 were found to be required for assembly of a stable diiron centre.

Crystal Structure Analysis of the Repair of Iron Centers Protein YtfE and Its Interaction with NO

“…[1,6] Recently,h omology studies proposed that the diiron centeri sc oordinated by H84, H129, H160, H204, E133, and E208. [7] Apo-YtfE is incapable of recovering damaged[ Fe-S] proteins in vitro, [2a] suggesting that the non-heme carboxylate-bridged diiron center is crucial for the YtfE activity.T he functionality of the YtfE redox states under nitrosative stress and its three-dimensional structure remain largely speculative.…”

The Di-iron RIC Protein (YtfE) of Escherichia coli Interacts with the DNA-Binding Protein from Starved Cells (Dps) To Diminish RIC Protein-Mediated Redox Stress

“…Fumarase activity of E. coli wild-type, Δric, Δdps, and Δdps Δric strains grown aerobically to an OD 600 of 0.6 (C). Complementation experiments of aconitase activity (D) were performed using Δric and Δdps Δric strains transformed with pUC18, and with pUC18 encoding the RIC protein and the mutated Glu133Leu-RIC (a RIC protein where glutamate 133 underwent site-directed mutation by leucine [10]). Values are represented as normalized to the aconitase or fumarase activity of wild-type cells.…”

“…BiFC assay was performed essentially as described previously (33). For this purpose, the genes encoding RIC a truncated version of the RIC (lacking the first 57 amino acid residues in the N terminal [10]), Dps, Bfr, and FtnA were PCR amplified from genomic DNA of E. coli K-12 using the oligonucleotides described in Table 2. The DNA fragments were cloned into vectors (pET11a-link-N-GFP and pMRBAD-link-C-GFP [33]) that express the green fluorescent protein (GFP) to allow formation of corresponding N-or C-terminal GFP fusions, respectively.…”

“…Prior to the activity assay, the cell pellets were resuspended in reaction buffer containing 0.5 mg/ml lysozyme and 0.2 mg/ml DNase, incubated on ice for 10 min, and then centrifuged at 9,600 ϫ g for 10 min at 4°C. The aconitase activity was determined in these supernatants (50 l) in reaction mixtures that also contained 200 M NADP ϩ , 1 U isocitrate dehydrogenase, and 30 mM sodium citrate (10), and by recording the formation of NADPH at 340 nm.…”

“…In particular, substitution of residues His129, Glu133, or Glu208 of the E. coli RIC abrogated its ability to protect the activity of aconitase. Moreover, two -carboxylate bridges contributed by Glu133 and Glu208, linking the two di-iron site atoms, were shown to be required for the assembly of a stable di-iron center (10). These studies also demonstrated the important contribution of the conserved His84, His129, His160, His204, Glu133, and Glu208 residues in ligating the di-iron center within the four-helix bundle fold, and these di-iron coordination roles were recently confirmed by X-ray crystallographic structural studies (11).…”