Iron-sulfur (Fe-S) clusters are key metal cofactors of metabolic, regulatory, and stress response proteins in most organisms. The unique properties of these clusters make them susceptible to disruption by iron starvation or oxidative stress. Both iron and sulfur can be perturbed under stress conditions, leading to Fe-S cluster defects. Bacteria and higher plants contain a specialized system for Fe-S cluster biosynthesis under stress, namely the Suf pathway. In Escherichia coli the Suf pathway consists of six proteins with functions that are only partially characterized. Here we describe how the SufS and SufE proteins interact with the SufBCD protein complex to facilitate sulfur liberation from cysteine and donation for Fe-S cluster assembly. It was previously shown that the cysteine desulfurase SufS donates sulfur to the sulfur transfer protein SufE. We have found here that SufE in turn interacts with the SufB protein for sulfur transfer to that protein. The interaction occurs only if SufC is present. Furthermore, SufB can act as a site for Fe-S cluster assembly in the Suf system. This provides the first evidence of a novel site for Fe-S cluster assembly in the SufBCD complex.Fe-S clusters perform important functions in multiple cellular processes, including respiration, gene regulation, and the trichloroacetic acid cycle (1). In vivo Fe-S biogenesis requires specific proteins such as a pyridoxal phosphate-dependent cysteine desulfurase that mobilizes sulfur from L-cysteine and an Fe-S cluster scaffold upon which the nascent cluster can be constructed prior to its transfer to an apoprotein. Homologues of these two core components can be found in numerous organisms ranging from bacteria to humans (1-3). In bacteria the basal pathway of Fe-S cluster assembly is known as Isc 5 (ironsulfur cluster assembly) (4, 5).Fe-S cluster homeostasis is sensitive to disruption by reactive oxygen and reactive nitrogen species or by iron limitation (6 -8). Recently it was discovered that biosynthesis of Fe-S clusters during adverse stress conditions such as iron starvation and oxidative stress requires a specialized assembly pathway different from the basal Isc system. This pathway, termed the Suf pathway (mobilization of sulfur), is regulated in response to those stress conditions (7-9). The Suf pathway is present in cyanobacteria and the chloroplast of higher plants as well as in bacteria, including human pathogens such as Yersinia pestis and Mycobacterium tuberculosis. In M. tuberculosis, the Suf pathway seems to be the primary Fe-S cluster assembly system, as deleting the suf genes is thought to be lethal to that organism (10).Because of its importance, Suf has been the focus of intense study at the biochemical level, especially in the Gram-negative bacterium Escherichia coli. The sufABCDSE operon in E. coli encodes six proteins. SufA is homologous to IscA in the basal Isc Fe-S cluster pathway. The exact function of SufA is unknown. Although it has been suggested to act as an Fe-S scaffold, it cannot be excluded that SufA pla...

Iron-sulfur (Fe-S) cluster-containing proteins are essential components of cells. In eukaryotes, Fe-S clusters are synthesized by the mitochondrial iron-sulfur cluster (ISC) machinery and the cytosolic iron-sulfur assembly (CIA) system. In the mammalian ISC machinery, preassembly of the Fe-S cluster on the scaffold protein (ISCU) involves a cysteine desulfurase complex (NFS1/ISD11) and frataxin (FXN), the protein deficient in Friedreich's ataxia. Here, by comparing the biochemical and spectroscopic properties of quaternary (ISCU/NFS1/ISD11/FXN) and ternary (ISCU/NFS1/ISD11) complexes, we show that FXN stabilizes the quaternary complex and controls iron entry to the complex through activation of cysteine desulfurization. Furthermore, we show for the first time that in the presence of iron and L-cysteine, an [Fe(4)S(4)] cluster is formed within the quaternary complex that can be transferred to mammalian aconitase (mACO2) to generate an active enzyme. In the absence of FXN, although the ternary complex can assemble an Fe-S cluster, the cluster is inefficiently transferred to ACO2. Taken together, these data help to unravel further the Fe-S cluster assembly process and the molecular basis of Friedreich's ataxia.