Rapid and quantitative reductive coupling of two 2Fe-2S2+ clusters to form a single 4Fe-4S2+ cluster on the homodimeric IscU Fe−S cluster scaffold protein has been demonstrated by UV−visible ...absorption, Mössbauer, and resonance Raman spectroscopies, using dithionite as the electron donor. Partial reductive coupling was also observed using reduced Isc ferredoxin, which raises the possibility that Isc ferredoxin is the physiological reductant. The results suggest that reductive coupling of adjacent 2Fe-2S2+ clusters assembled on IscU provides a general mechanism for the final step in the biosynthesis of 4Fe-4S2+ clusters. The 4Fe-4S2+ center on IscU can be reduced to a S = 1/24Fe-4S+ cluster (g ∥ = 2.06 and g ⊥ = 1.92), but the low midpoint potential (< −570 mV) and instability of the reduced cluster argue against any physiological relevance for the reduced cluster. On exposure to O2, the 4Fe-4S2+ cluster on IscU degrades via a semistable 2Fe-2S2+ cluster with properties analogous to those of the 2Fe-2S2+ center in 2Fe-2S2+ IscU. It is suggested that the ability of IscU to accommodate either 2Fe-2S2+ or 4Fe-4S2+ clusters in response to cellular redox status and/or oxygen levels may provide an effective way to populate appropriately cluster-loaded forms of IscU for maturation of different types of Fe−S proteins.
The transcription of iron uptake and storage genes in Saccharomyces cerevisiae is primarily regulated by the transcription factor Aft1. Nucleocytoplasmic shuttling of Aft1 is dependent upon ...mitochondrial Fe-S cluster biosynthesis via a signaling pathway that includes the cytosolic monothiol glutaredoxins (Grx3 and Grx4) and the BolA homologue Fra2. However, the interactions between these proteins and the iron-dependent mechanism by which they control Aft1 localization are unclear. To reconstitute and characterize components of this signaling pathway in vitro, we have overexpressed yeast Fra2 and Grx3/4 in Escherichia coli. We have shown that coexpression of recombinant Fra2 with Grx3 or Grx4 allows purification of a stable 2Fe-2S2+ cluster-containing Fra2-Grx3 or Fra2-Grx4 heterodimeric complex. Reconstitution of a 2Fe-2S cluster on Grx3 or Grx4 without Fra2 produces a 2Fe-2S-bridged homodimer. UV−visible absorption and CD, resonance Raman, EPR, ENDOR, Mössbauer, and EXAFS studies of 2Fe-2S Grx3/4 homodimers and the 2Fe-2S Fra2-Grx3/4 heterodimers indicate that inclusion of Fra2 in the Grx3/4 Fe-S complex causes a change in the cluster stability and coordination environment. Taken together, our analytical, spectroscopic, and mutagenesis data indicate that Grx3/4 and Fra2 form a Fe-S-bridged heterodimeric complex with Fe ligands provided by the active site cysteine of Grx3/4, glutathione, and a histidine residue. Overall, these results suggest that the ability of the Fra2-Grx3/4 complex to assemble a 2Fe-2S cluster may act as a signal to control the iron regulon in response to cellular iron status in yeast.
Saccharomyces cerevisiae mitochondrial glutaredoxin 5 (Grx5) is the archetypical member of a ubiquitous class of monothiol glutaredoxins with a strictly conserved CGFS active-site sequence that has ...been shown to function in biological Fe2S2(2+) cluster trafficking. In this work, we show that recombinant S. cerevisiae Grx5 purified aerobically, after prolonged exposure of the cell-free extract to air or after anaerobic reconstitution in the presence of glutathione, predominantly contains a linear Fe3S4(+) cluster. The excited-state electronic properties and ground-state electronic and vibrational properties of the linear Fe3S4(+) cluster have been characterized using UV-vis absorption/CD/MCD, EPR, Mossbauer, and resonance Raman spectroscopies. The results reveal a rhombic S = 5/2 linear Fe3S4(+) cluster with properties similar to those reported for synthetic linear Fe3S4(+) clusters and the linear Fe3S4(+) clusters in purple aconitase. Moreover, the results indicate that the Fe-S cluster content previously reported for many monothiol Grxs has been misinterpreted exclusively in terms of Fe2S2(2+) clusters, rather than linear Fe3S4(+) clusters or mixtures of linear Fe3S4(+) and Fe2S2(2+) clusters. In the absence of GSH, anaerobic reconstitution of Grx5 yields a dimeric form containing one Fe4S4(2+) cluster that is competent for in vitro activation of apo-aconitase, via intact cluster transfer. The ligation of the linear Fe3S4(+) and Fe4S4(2+) clusters in Grx5 has been assessed by spectroscopic, mutational, and analytical studies. Potential roles for monothiol Grx5 in scavenging and recycling linear Fe3S4(+) clusters released during protein unfolding under oxidative stress conditions and in maturation of Fe4S4(2+) cluster-containing proteins are discussed in light of these results.
Iron-sulfur clusters (Fe-S clusters) are assembled on molecular scaffolds and subsequently used for maturation of proteins that require Fe-S clusters for their functions. Previous studies have shown ...that Azotobacter vinelandii produces at least two Fe-S cluster assembly scaffolds: NifU, required for the maturation of nitrogenase, and IscU, required for the general maturation of other Fe-S proteins. A. vinelandii also encodes a protein designated NfuA, which shares amino acid sequence similarity with the C-terminal region of NifU. The activity of aconitase, a 4Fe-4S cluster-containing enzyme, is markedly diminished in a strain containing an inactivated nfuA gene. This inactivation also results in a null-growth phenotype when the strain is cultivated under elevated oxygen concentrations. NifU has a limited ability to serve the function of NfuA, as its expression at high levels corrects the defect of the nfuA-disrupted strain. Spectroscopic and analytical studies indicate that one 4Fe-4S cluster can be assembled in vitro within a dimeric form of NfuA. The resultant 4Fe-4S cluster-loaded form of NfuA is competent for rapid in vitro activation of apo-aconitase. Based on these results a model is proposed where NfuA could represent a class of intermediate Fe-S cluster carriers involved in Fe-S protein maturation.
The reaction of colorless iron(II) formate or the mixed-valence cluster Fe(3)O(MeCOO)(6)(H(2)O)(3) with formic acid in dimethylformamide exposed to air at 110 degrees C affords black crystals of the ...mixed-valence (Me(2)NH(2))Fe(II)Fe(III)(HCOO)(6) three-dimensional (3D) structure in which the cations occupy half of the channels. The structure consists of alternating layers of Fe(II)O(6) Fe(1)-O(1), 2.119(1) A and Fe(III)O(6) Fe(2)-O(2), 2.0049(9) A octahedra bridged by anti-anti-bonded formates to afford an open-framework 3D structure. The structure is very similar to those of (Me(2)NH(2))Fe(II)(HCOO)(3) and Fe(III)(HCOO)(3).HCOOH, both of which are colorless. The black crystals appear dark-purple (lambda(max) approximately 520 nm) when powdered. The room-temperature Mössbauer spectrum confirms the 1:1 ratio of Fe(II) (delta = 1.03 mm/s, DeltaE(Q) = 1.16 mm/s) and Fe(III) (delta = 0.62 mm/s, DeltaE (Q) = 0.49 mm/s). Magnetic ordering that includes negative magnetization at low fields occurs at low temperature. The only molecular-based magnetic materials in which this phenomenon has been observed are the 2D polyiron(II,III) oxalates AFe(II)Fe(III)(C(2)O(4))(3) (A = R(4)N(+) cation).
Glutaredoxins (Grxs) are small oxidoreductases that reduce disulphide bonds or protein‐glutathione mixed disulphides. More than 30 distinct grx genes are expressed in higher plants, but little is ...currently known concerning their functional diversity. This study presents biochemical and spectroscopic evidence for incorporation of a 2Fe–2S cluster in two heterologously expressed chloroplastic Grxs, GrxS14 and GrxS16, and in vitro cysteine desulphurase‐mediated assembly of an identical 2Fe–2S cluster in apo‐GrxS14. These Grxs possess the same monothiol CGFS active site as yeast Grx5 and both were able to complement a yeast grx5 mutant defective in Fe–S cluster assembly. In vitro kinetic studies monitored by CD spectroscopy indicate that 2Fe–2S clusters on GrxS14 are rapidly and quantitatively transferred to apo chloroplast ferredoxin. These data demonstrate that chloroplast CGFS Grxs have the potential to function as scaffold proteins for the assembly of 2Fe–2S clusters that can be transferred intact to physiologically relevant acceptor proteins. Alternatively, they may function in the storage and/or delivery of preformed Fe–S clusters or in the regulation of the chloroplastic Fe–S cluster assembly machinery.
Nfu-type proteins are essential in the biogenesis of iron–sulfur (Fe-S) clusters in numerous organisms. A number of phenotypes including low levels of Fe-S cluster incorporation are associated with ...the deletion of the gene encoding a chloroplast-specific Nfu-type protein, Nfu2 from Arabidopsis thaliana (AtNfu2). Here, we report that recombinant AtNfu2 is able to assemble both 2Fe-2S and 4Fe-4S clusters. Analytical data and gel filtration studies support cluster/protein stoichiometries of one 2Fe-2S cluster/homotetramer and one 4Fe-4S cluster/homodimer. The combination of UV–visible absorption and circular dichroism and resonance Raman and Mössbauer spectroscopies has been employed to investigate the nature, properties, and transfer of the clusters assembled on Nfu2. The results are consistent with subunit-bridging 2Fe-2S2+ and 4Fe-4S2+ clusters coordinated by the cysteines in the conserved CXXC motif. The results also provided insight into the specificity of Nfu2 for the maturation of chloroplastic Fe-S proteins via intact, rapid, and quantitative cluster transfer. 2Fe-2S cluster-bound Nfu2 is shown to be an effective 2Fe-2S2+ cluster donor for glutaredoxin S16 but not glutaredoxin S14. Moreover, 4Fe-4S cluster-bound Nfu2 is shown to be a very rapid and efficient 4Fe-4S2+ cluster donor for adenosine 5′-phosphosulfate reductase (APR1), and yeast two-hybrid studies indicate that APR1 forms a complex with Nfu2 but not with Nfu1 and Nfu3, the two other chloroplastic Nfu proteins. This cluster transfer is likely to be physiologically relevant and is particularly significant for plant metabolism as APR1 catalyzes the second step in reductive sulfur assimilation, which ultimately results in the biosynthesis of cysteine, methionine, glutathione, and Fe-S clusters.
Genetic experiments have established that IscU is involved in maturation of Fe−S proteins that require either 2Fe-2S or 4Fe-4S clusters for their biological activities. Biochemical studies have also ...shown that one 2Fe-2S cluster can be assembled in vitro within each subunit of the IscU homodimer and that these clusters can be reductively coupled to form a single 4Fe-4S cluster. In the present work, it is shown that the 4Fe-4S cluster-loaded form of A. vinelandii IscU, but not the 2Fe-2S cluster-loaded form, can be used for intact cluster transfer to an apo form of A. vinelandii aconitase A, a member of the monomeric dehydratase family of proteins that requires a 4Fe-4S cluster for enzymatic activity. The rate of 4Fe-4S cluster transfer from IscU to apo-aconitase A was not affected by the presence of the HscA/HscB co-chaperone system and MgATP. However, an altered form of a 4Fe-4S cluster-containing IscU, having the highly conserved aspartate-39 residue substituted with alanine, is an effective inhibitor of wild-type 4Fe-4S cluster-loaded IscU-directed activation of apo-aconitase A. In contrast, neither the clusterless form of IscU nor the 2Fe-2S cluster-loaded form of IscU is an effective inhibitor of IscU-directed apo-aconitase A activation. These results are interpreted to indicate that the 2Fe-2S and 4Fe-4S cluster-loaded forms of IscU adopt different conformations that provide specificity with respect to the maturation of 2Fe-2S and 4Fe-4S centers in proteins.
We report the generation and characterization of a diiron(III) intermediate formed during reaction with dioxygen of the reduced hydroxylase component of toluene/o-xylene monooxygenase from ...Pseudomonas sp. OX1. The decay rate of this species is accelerated upon mixing with phenol, a substrate for this system. Under steady-state conditions, hydrogen peroxide was generated in the absence of substrate. The oxidized hydroxylase also decomposed hydrogen peroxide to liberate dioxygen in the absence of reducing equivalents. This activity suggests that dioxygen activation may be reversible. The linear free energy relationship determined from hydroxylation of para-substituted phenols under steady-state turnover has a negative slope. A value of ρ < 0 is consistent with electrophilic attack by the oxidizing intermediate on the aromatic substrates. The results from these steady and pre-steady-state experiments provide compelling evidence that the diiron(III) intermediate is the active oxidant in the toluene/o-xylene monooxygenase system and is a peroxodiiron(III) transient, despite differences between its optical and Mössbauer spectroscopic parameters and those of other peroxodiiron(III) centers.
Iron-sulfur (Fe-S) clusters serve as cofactors in many proteins that have important redox, catalytic, and regulatory functions. In bacteria, biogenesis of Fe-S clusters is mediated by multiple gene ...products encoded by the isc and nif operons. In particular, genetic and biochemical studies suggest that IscU, Nfu, and IscA function as scaffold proteins for assembly and delivery of rudimentary Fe-S clusters to target proteins. Here we report the characterization of human Nfu. A combination of biochemical and spectroscopic techniques, including UV-visible absorption and$^{57}Fe\>M\ddot ossbauer$spectroscopies, have been used to investigate the ability of purified human Nfu to assemble Fe-S clusters. The results suggest that Nfu can assemble approximately one labile 4Fe-4S cluster per two Nfu monomers, and support the proposal that Nfu is an alternative scaffold protein for assembly of clusters that are subsequently used for maturation of targeted Fe-S proteins. Analyses of genomic DNA, transcripts, and translation products indicate that alternative splicing of a common pre-mRNA results in synthesis of two Nfu isoforms with distinct subcellular localizations. Isoform I is localized in the mitochondria, whereas isoform II is present in the cytosol and the nucleus. These results, together with previous reports of subcellular distributions of isoforms of human IscS and IscU in mitochondria, cytosol, and nucleus suggest that the Fe-S cluster assembly machineries are compartmentalized in higher eukaryotes.
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