The scaffold protein IscU and molecular chaperones HscA and HscB play central roles in biological assembly of iron-sulfur clusters and maturation of iron-sulfur proteins. However, the structure of IscU-FeS complexes and the molecular mechanism whereby the chaperones facilitate cluster transfer to acceptor proteins are not well understood. We have prepared amino acid substitution mutants of Escherichia coli IscU in which potential ligands to the FeS cluster (Cys-37, Cys-63, His-105, and Cys-106) were individually replaced with alanine. The properties of the IscU-FeS complexes formed were investigated by measuring both their ability to transfer preformed FeS clusters to apo-ferredoxin and the activity of the IscU proteins in catalyzing cluster assembly on apo-ferredoxin using inorganic iron with inorganic sulfide or with IscS and cysteine as a sulfur source. The ability of the HscA/HscB chaperone system to accelerate ATP-dependent cluster transfer from each IscU substitution mutant to apo-ferredoxin was also determined. All of the mutants formed FeS complexes with a stoichiometry similar to the wild-type holo-protein, i.e., IscU(2)[2Fe2S], raising the possibility that different cluster ligation states may occur during iron-sulfur protein maturation. Spectroscopic properties of the mutants and the kinetics of transfer of performed IscU-FeS clusters to apo-ferredoxin indicate that the most stable form of holo-IscU involves iron coordination by Cys-63 and Cys-106. Results of studies on the ability of mutants to catalyze formation of holo-ferredoxin using iron and different sulfur sources were consistent with proposed roles for Cys-63 and Cys-106 in FeS cluster binding and also indicated an essential role for Cys-106 in sulfide transfer to IscU from IscS. Measurements of the ability of the chaperones HscA and HscB to facilitate cluster transfer from holo-IscU to apo-ferredoxin showed that only IscU(H105A) behaved similarly to wild-type IscU in exhibiting ATP-dependent stimulation of cluster transfer. IscU(C63A) and IscU(C106A) displayed elevated rates of cluster transfer in the ±ATP whereas IscU(C37A) exhibited low rates of cluster transfer ±ATP. In interpreting these findings, we propose that IscU(2)[2Fe2S] is able undergo structural isomerization to yield conformers having different cysteine residues bound to the cluster. On the basis of the crystal structure of HscA complexed with an IscU-derived peptide, we propose that the chaperone binds and stabilizes an isomer of IscU(2)[2Fe2S] in which the cluster is bound by cysteine residues 37 and 63 and that the [2Fe2S] cluster, being held less tightly than that coordinated by Cys-63 and Cys-106 in free IscU(2)[2Fe2S], is more readily transferred to acceptor proteins such as apo-ferredoxin.
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.
The relationship between iron uptake by aporubredoxins (apoRds) and formation of native holorubredoxins (holoRd), including their Fe(SCys) 4 sites, was studied. In the absence of denaturants, apoRds exhibited spectroscopic features consistent with structures very similar to those of the folded holoRds. However, additions of either ferric or ferrous salts to the apoRds in the absence of denaturants gave less than 40% recovery of the native holoRd circular dichroism and UV-vis spectroscopic features. In the presence of either 6 M urea or 6 M guanidine hydrochloride, the nativelike structural features of the apoRds were absent. Nevertheless, nearly quantitative recoveries of the native holoRd spectroscopic features were achieved by addition of either ferric or ferrous salts to the denatured apoRds without diluting the denaturant. Consistent with this observation, the native spectroscopic features were unaffected by addition of the same denaturant concentrations to the as-isolated holoRds. Denaturing concentrations of urea or guanidine hydrochloride also increased the rates of holoRd recoveries from apoRds and ferrous salts. Mass spectrometry confirmed that ferric iron binding to the denatured apoRds precedes the recoveries of protein secondary structures and Fe(SCys) 4 sites. Thus, iron binding to the apoRds guides, both kinetically and thermodynamically, refolding to the native holoRd structures. Our results imply that the ferrous oxidation state would more efficiently drive formation of the native holoRd structure from the nascent apoprotein in vivo, but that the Fe(SCys) 4 site must attain the ferric state in order to achieve its native structure.Correspondence to: Francesco Bonomi. Electronic supplementary material The online version of this article
Addition of iron salts to chaotrope-denatured aporubredoxin (apoRd) leads to nearly quantitative recovery of its single Fe(SCys) 4 site and native protein structure without significant dilution of the chaotrope. This "high chaotrope" approach was used to examine iron binding and protein folding events using stopped-flow UV/vis absorption and CD spectroscopies. At 100-fold molar excess ferrous iron over denatured apoRd maintained in 5 M urea, the folded holoFe III Rd structure was recovered in >90% yield with t 1/2 < 10 msec. More modest excesses of iron also gave nearly quantitative holoRd formation in 5 M urea but with chronological resolution of iron binding and protein folding events. The results indicate structural recovery in 5 M urea consists of the minimal sequence: (1) binding of ferrous iron to the unfolded apoRd, (2) rapid formation of a near-native ferrous Fe(SCys) 4 site within a protein having no detectable secondary structure, (3) recovery of the ferrous Fe(SCys) 4 site chiral environment nearly concomitantly with (4) recovery of the native protein secondary structure. The rate of step 2 (and by inference, step 1) was not saturated even at 100-fold molar excess of iron. Analogous results obtained on Cys→Ser iron ligand variants support formation of an unfolded-Fe(SCys) 3 complex between steps 1 and 2, which we propose is the key nucleation event that pulls distal regions of the protein chain together. These results show that folding of chaotrope-denatured apoRd is iron-nucleated and driven by extraordinarily rapid formation of the Fe(SCys) 4 site from an essentially random coil apoprotein. This high chaotrope, multi-spectroscopic approach could clarify folding pathways of other [M(SCys) 3 Supporting Information AvailableNear UV-CD spectra of apo and holoRds in high urea, plots of midpoint urea denaturations of apoRds, spectral time courses of ferrous ammonium sulfate oxidations in 5 M urea, static far-UV CD spectra of apo and holoRds in high urea, semi-log plots of the time courses in Figure 3 and Figure 6, stopped-flow absorption spectral time course for apoRd +110-fold molar excess iron in 4.6 M urea. This material is available free of charge via the Internet at http://pubs.acs.org. 1 Abbreviations used: Rd, rubredoxin, apoRd, metal-free Rd; holoRd, iron-containing Rd in its native folded structure; Cp, Clostridium pasteurianum; CD, circular dichroism; wt, wild type; Tris-HCl, tris(hydroxymethyl)aminomethane-hydrochloride; CXXC, two cysteine residues (C) separated by two other residues, X, in the Rd amino acid sequence; C6S, C9S, C39S, C42S, Cys→Ser variant Rds; unfolded-Fe II (SCys) 4 , species exhibiting near-UV absorption characteristic of the native Fe II (SCys) 4 site but no CD signal for protein secondary structure; chiral-Fe II (SCys) 4 , species exhibiting near-UV absorption and CD signals characteristic of the native Fe II (SCys) 4 site; folded-Fe II (SCys) 4 , species exhibiting absorption and CD signals for the Fe II (SCys) 4 site and protein secondary structure CD signal char...
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