Role of IscX in Iron-Sulfur Cluster Biogenesis in Escherichia coli

Journal Article · Wed Aug 20 00:00:00 EDT 2014 · J. Am. Chem. Soc.

DOI:https://doi.org/10.1021/ja501260h· OSTI ID:1140207

Kim, Jin Hae; Bothe, Jameson R.; Frederick, Ronnie O.; Holder, Johneisa C.; Markley, John L. ^[1]

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1. UW

The Escherichia coli isc operon encodes key proteins involved in the biosynthesis of iron–sulfur (Fe–S) clusters. Whereas extensive studies of most ISC proteins have revealed their functional properties, the role of IscX (also dubbed YfhJ), a small acidic protein encoded by the last gene in the operon, has remained in question. Previous studies showed that IscX binds iron ions and interacts with the cysteine desulfurase (IscS) and the scaffold protein for cluster assembly (IscU), and it has been proposed that IscX functions either as an iron supplier or a regulator of Fe–S cluster biogenesis. We have used a combination of NMR spectroscopy, small-angle X-ray scattering (SAXS), chemical cross-linking, and enzymatic assays to enlarge our understanding of the interactions of IscX with iron ions, IscU, and IscS. We used chemical shift perturbation to identify the binding interfaces of IscX and IscU in their complex. NMR studies showed that Fe²⁺ from added ferrous ammonium sulfate binds IscX much more avidly than does Fe³⁺ from added ferric ammonium citrate and that Fe²⁺ strengthens the interaction between IscX and IscU. We found that the addition of IscX to the IscU–IscS binary complex led to the formation of a ternary complex with reduced cysteine desulfurase activity, and we determined a low-resolution model for that complex from a combination of NMR and SAXS data. We postulate that the inhibition of cysteine desulfurase activity by IscX serves to reduce unproductive conversion of cysteine to alanine. By incorporating these new findings with results from prior studies, we propose a detailed mechanism for Fe–S cluster assembly in which IscX serves both as a donor of Fe²⁺ and as a regulator of cysteine desulfurase activity.

• Research Organization: Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
• Sponsoring Organization: OTHERDOE - BASIC ENERGY SCIENCESNIH
• OSTI ID: 1140207
• Journal Information: J. Am. Chem. Soc., Vol. 136, Issue (22) ; 06, 2014
• Country of Publication: United States
• Language: ENGLISH