Iron-Sulfur Cluster Assembly: CHARACTERIZATION OF IscA AND EVIDENCE FOR A SPECIFIC AND FUNCTIONAL COMPLEX WITH FERREDOXIN

Sandrine Ollagnier-De-Choudens,Tony Mattioli,Yasuhiro Takahashi,Marc Fontecave

doi:10.1074/jbc.m102902200

Abstract

The synthesis of iron-sulfur clusters in Escherichia coli is believed to require a complex protein machinery encoded by the isc (iron-sulfur cluster) operon. The product of one member of this operon, IscA, has been overexpressed, purified, and characterized. It can assemble an air-sensitive [2Fe-2S] cluster as shown by UV-visible and resonance Raman spectroscopy. The metal form but not the apoform of IscA binds ferredoxin, another member of the isc operon, selectively, allowing transfer of iron and sulfide from IscA to ferredoxin and formation of the [2Fe-2S] holoferredoxin. These results thus suggest that IscA is involved in ferredoxin cluster assembly and activation. This is an important function because a functional ferredoxin is required for maturation of other cellular Fe-S proteins.

Highlights

The synthesis of iron-sulfur clusters in Escherichia coli is believed to require a complex protein machinery encoded by the isc operon
It has been recently shown that E. coli IscA greatly improves the efficiency of the maturation of overexpressed ferredoxins within E. coli cells [7]
These results have suggested that these cysteines constitute an iron binding motif and that IscA proteins participate in Fe-S cluster assembly through the binding of an intermediate Fe-S cluster that is subsequently transferred to target polypeptides

Summary

EXPERIMENTAL PROCEDURES

Expression and Purification—E. coli C41(DE3)pET21-EcFdx and C41(DE3)pRKISC cells for expression of Fdx and the products of the isc operon, respectively, were grown as described by Nakamura et al [21]. The iscA gene was cloned into BamHI-SalI sites of pQE-30 (Qiagen) and pMAL-c2 (New England BioLabs, Inc.) vectors to make pQiscA-55 and pMiscA-15 for production of IscA either with a histidine tag (IscAH) or fused with the maltose-binding protein (MBP-IscA), respectively. E. coli M15(pREP4) (Qiagen) and TB1 (New England BioLabs, Inc.) strains were transformed with pQiscA-55 and pMiscA-15, respectively. M15(pREP4)pQiscA-55 cells (17 g) were sonicated in buffer A (20 mM sodium phosphate, pH 7.4, 50 mM NaCl) containing approximately 20 ␮g of DNase I, 10 mM MgCl2, and protease inhibitors (1 tablet, Roche Molecular Biochemicals), and after centrifugation (90 min, 45,000 rpm, 5 °C) soluble proteins (1.2 g) were used for further purification. TB1pMiscA-15 cells (16 g) were sonicated in buffer containing 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 1 mM EDTA, and 1 mM dithiothreitol and centrifuged (90 min, 45,000 rpm, 5 °C). Iron and Sulfide Binding to IscA—The protein was incubated for 3 h inside a glove box (Jacomex B553 (NMT)) with a 3–5-fold molar excess of both Na2S (Fluka) and Fe(NH4)2(SO4) (Aldrich) in the presence of 5 mM dithiothreitol in 0.1 M Tris-HCl, pH 8.0, and desalted on Sephadex G-25 (80 ml, same buffer)

The abbreviations used are

RESULTS

DISCUSSION