Purified sakacin A shows a dual mechanism of action against Listeria spp: proton motive force dissipation and cell wall breakdown.

Sakacin A was purified to homogeneity through simple chromatographic procedures from cultures of Lactobacillus sakei DSMZ 6333 grown on a low-cost medium. The highly purified protein dissipated both transmembrane potential (ΔΨ) and transmembrane pH gradient (ΔpH) in Listeria cells in a very intense, rapid, and energy-dependent fashion. On a slower timescale, purified sakacin A also showed a lytic activity toward isolated cell walls of Listeria. Mass spectrometry was used to analyze the products of sakacin A action on cell walls, evidencing that sakacin A acts on various types of bonds within peptoglycans.

Facilitated transfer of IscU-[2Fe2S] clusters by chaperone-mediated ligand exchange.

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.

Functional characterization of Fdx1: evidence for an evolutionary relationship between P450-type and ISC-type ferredoxins.

Ferredoxins are ubiquitous proteins with electron transfer activity involved in a variety of biological processes. In this work, we investigated the characteristics and function of Fdx1 from Sorangium cellulosum So ce56 by using a combination of bioinformatics and of biochemical/biophysical approaches. We were able to experimentally confirm a role of Fdx1 in the iron-sulfur cluster biosynthesis by in vitro reduction studies with cluster-loaded So ce56 IscU and by transfer studies of the cluster from the latter protein to apo-aconitase A. Moreover, we found that Fdx1 can replace mammalian adrenodoxin in supporting the activity of bovine CYP11A1. This makes S. cellulosum Fdx1 the first prokaryotic ferredoxin reported to functionally interact with this mammalian enzyme. Although the interaction with CYP11A1 is non-physiological, this is-to the best of our knowledge-the first study to experimentally prove the activity of a postulated ISC-type ferredoxin in both the ISC assembly and a cytochrome P450 system. This proves that a single ferredoxin can be structurally able to provide electrons to both cytochromes P450 and IscU and thus support different biochemical processes. Combining this finding with phylogenetic and evolutionary trace analyses led us to propose the evolution of eukaryotic mitochondrial P450-type ferredoxins and ISC-type ferredoxins from a common prokaryotic ISC-type ancestor.

Iron-nucleated folding of a metalloprotein in high urea: resolution of metal binding and protein folding events.

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. With a 100-fold molar excess of ferrous iron over denatured apoRd maintained in 5 M urea, the folded holoFe(III)Rd structure was recovered in >90% yield with a t(1/2) of <10 ms. 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, and (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 a 100-fold molar excess of iron. Analogous results obtained for 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 together distal regions of the protein chain. 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, multispectroscopy approach could clarify folding pathways of other [M(SCys)(3)]- or [M(SCys)(4)]-containing proteins.

Studies on the mechanism of catalysis of iron-sulfur cluster transfer from IscU[2Fe2S] by HscA/HscB chaperones.

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 chaperone-catalyzed 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 --> R transition of HscA accompanying ATP hydrolysis.

"Iron priming" guides folding of denatured aporubredoxins.

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.

Contribution of the [FeII(SCys)4] site to the thermostability of rubredoxins.

The thermostabilities of Fe(2+) ligation in rubredoxins (Rds) from the hyperthermophile Pyrococcus furiosus (Pf) and the mesophiles Clostridium pasteurianum (Cp) and Desulfovibrio vulgaris (Dv) were compared. Residue 44 forms an NH.S(Cys) hydrogen bond to one of the cysteine ligands to the [Fe(SCys)(4)] site, and substitutions at this location affect the redox properties of the [Fe(SCys)(4)] site. Both Pf Rd and Dv Rd have an alanine residue at position 44, whereas Cp Fd has a valine residue. Wild-type proteins were examined along with V44A and A44V "exchange" mutants of Cp and Pf Rds, respectively, in order to assess the effects of the residue at position 44 on the stability of the [Fe(SCys)(4)] site. Stability of iron ligation was measured by temperature-ramp and fixed-temperature time course experiments, monitoring iron release in both the absence and presence of more thiophilic metals (Zn(2+), Cd(2+)) and over a range of pH values. The thermostability of the polypeptide fold was concomitantly measured by fluorescence, circular dichroism, and (1)H NMR spectroscopies. The A44V mutation strongly lowered the stability of the [Fe(II)(SCys)(4)] site in Pf Rd, whereas the converse V44A mutation of Cp Rd significantly raised the stability of the [Fe(II)(SCys)(4)] site, but not to the levels measured for wild-type Dv Rd. The region around residue 44 is thus a significant contributor to stability of iron coordination in reduced Rds. This region, however, made only a minor contribution to the thermostability of the protein folding, which was found to be higher for hyperthermophilic versus mesophilic Rds, and largely independent of the residue at position 44. These results, together with our previous studies, show that localized charge density, solvent accessibility, and iron site/backbone interactions control the thermostability of the [Fe(SCys)(4)] site. The iron site thermostability does make a minor contribution to the overall Rd thermostability. From a mechanistic standpoint, we also found that attack of displacing ions (H(+), Cd(2+)) on the Cys42 sulfur ligand at the [Fe(SCys)(4)] site occurs through the V8 side and not the V44 side of the iron site.