Studies on the Mechanism of Catalysis of Iron−Sulfur Cluster Transfer from IscU[2Fe2S] by HscA/HscB Chaperones

Abstract

The HscA/HscB chaperone/cochaperone system accelerates transfer of iron-sulfur clusters from the FeS-scaffold protein IscU (IscU 2 [2Fe2S], holo-IscU) to acceptor proteins in an ATP-dependent manner. We have employed visible region circular dichroism (CD) measurements to monitor chaperonecatalyzed cluster transfer from holo-IscU to apoferredoxin and to investigate chaperone-induced changes in properties of the IscU 2 [2Fe2S] cluster. HscA-mediated acceleration of [2Fe2S] cluster transfer exhibited an absolute requirement for both HscB and ATP. A mutant form of HscA lacking ATPase activity, HscA(T212V), was unable to accelerate cluster transfer, suggesting that ATP hydrolysis and conformational changes accompanying the ATP (T-state) to ADP (R-state) transition in the HscA chaperone are required for catalysis. Addition of HscA and HscB to IscU 2 [2Fe2S] did not affect the properties of the [2Fe2S] cluster, but subsequent addition of ATP was found to cause a transient change of the visible region CD spectrum, indicating distortion of the IscU-bound cluster. The dependence of the rate of decay of the observed CD change on ATP concentration and the lack of an effect of the HscA(T212V) mutant were consistent with conformational changes in the cluster coupled to ATP hydrolysis by HscA. Experiments carried out under conditions with limiting concentrations of HscA, HscB, and ATP further showed that formation of a 1:1:1 HscA-HscB-IscU 2 [2Fe2S] complex and a single ATP hydrolysis step are sufficient to elicit the full effect of the chaperones on the [2Fe2S] cluster. These results suggest that acceleration of iron-sulfur cluster transfer involves a structural change in the IscU 2 [2Fe2S] complex during the T w R transition of HscA accompanying ATP hydrolysis.

Global Identification of Genes Affecting Iron-Sulfur Cluster Biogenesis and Iron Homeostasis

“…Nevertheless, the effect of the deficiency of atpABCEFGH on Fe-S cluster biogenesis and iron homeostasis appears more significant than its effect on growth. ATP is required to generate a driving force for the CydD glutathione transporter (76,77) and for HscA to transfer a preformed Fe-S cluster to various target apo-proteins (50)(51)(52), consistent with the result that impaired ATP biosynthesis in the atp mutants results in decreased Fe-S enzyme activities. Thus, Fe-S cluster maturation and/or iron homeostasis may be strictly influenced by ATP availability (Fig.…”

“…The scaffold protein IscU receives sulfur directly from the sulfane sulfur on IscS (46,47) to form a [2Fe-2S]/[4Fe-4S] cluster in vitro (48,49). It is proposed that the Fe-S cluster assembled on the IscU scaffold can be incorporated into target proteins with the help of the DnaKJ-like proteins HscA and HscB (50)(51)(52). Analysis of the solution structure has revealed that apo-IscU exists in two different conformations, one structured and the other disordered, which can interconvert (53,54).…”

Functional reconstitution of mitochondrial Fe/S cluster synthesis on Isu1 reveals the involvement of ferredoxin

“…36). The Saccharomyces cerevisiae cysteine desulphurase complex Nfs1-Isd11, frataxin Yfh1, ferredoxin Yah1 and the scaffold Isu1 were overproduced and purified from E. coli ( Supplementary Fig.…”

The Role of Mitochondria in Cellular Iron-Sulfur Protein Biogenesis: Mechanisms, Connected Processes, and Diseases

“…The released Fe/S cluster may then be transferred to the mitochondrial monothiol glutaredoxin Grx5 or directly to apoproteins (Chandramouli and Johnson 2006;Bonomi et al 2008). Grx5 possibly serves as an Fe/S cluster transfer protein by transiently coordinating the Isu1-derived [2Fe-2S] cluster between two protein subunits and two protein-bound glutathione molecules (Bandyopadhyay et al 2008b;Shakamuri et al 2012).…”