IscU as a scaffold for iron-sulfur cluster biosynthesis: sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU

Iron-sulfur cluster biosynthesis in both prokaryotic and eukaryotic cells is known to be mediated by two highly conserved proteins, termed IscS and IscU in prokaryotes. The homodimeric IscS protein has been shown to be a cysteine desulfurase that catalyzes the reductive conversion of cysteine to alanine and sulfide. In this work, the time course of IscS-mediated Fe-S cluster assembly in IscU was monitored via anaerobic anion exchange chromatography. The nature and properties of the clusters assembled in discrete fractions were assessed via analytical studies together with absorption, resonance Raman, and M?ssbauer investigations. The results show sequential cluster assembly with the initial IscU product containing one [2Fe-2S](2+) cluster per dimer converting first to a form containing two [2Fe-2S](2+) clusters per dimer and finally to a form that contains one [4Fe-4S](2+) cluster per dimer. Both the [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU are reductively labile and are degraded within minutes upon being exposed to air. On the basis of sequence considerations and spectroscopic studies, the [2Fe-2S](2+) clusters in IscU are shown to have incomplete cysteinyl ligation. In addition, the resonance Raman spectrum of the [4Fe-4S](2+) cluster in IscU is best interpreted in terms of noncysteinyl ligation at a unique Fe site. The ability to assemble both [2Fe-2S](2+) and [4Fe-4S](2+) clusters in IscU supports the proposal that this ubiquitous protein provides a scaffold for IscS-mediated assembly of clusters that are subsequently used for maturation of apo Fe-S proteins.     

The catalytic subunit of Escherichia coli nitrate reductase A contains a novel [4Fe-4S] cluster with a high-spin ground state

We have used EPR spectroscopy, redox potentiometry, and protein crystallography to characterize the [4Fe-4S] cluster (FS0) of the Escherichia coli nitrate reductase A (NarGHI) catalytic subunit (NarG). FS0 is clearly visible in the crystal structure of NarGHI [Bertero, M. G., et al. (2003) Nat. Struct. Biol. 10, 681-687] but has novel coordination comprising one His residue and three Cys residues. At low temperatures (<15 K), reduced NarGHI exhibits a previously unobserved EPR signal comprising peaks at g = 5.023 and g = 5.556. We have assigned these features to a [4Fe-4S](+) cluster with an S = (3)/(2) ground state, with the g = 5.023 and g = 5.556 peaks corresponding to subpopulations exhibiting DeltaS = (1)/(2) and DeltaS = (3)/(2) transitions, respectively. Both peaks exhibit midpoint potentials of approximately -55 mV at pH 8.0 and are eliminated in the EPR spectrum of apomolybdo-NarGHI. The structure of apomolybdo-NarGHI reveals that FS0 is still present but that there is significant conformational disorder in a segment of residues that includes one of the Cys ligands. On the basis of these observations, we have assigned the high-spin EPR features of reduced NarGHI to FS0.     

Subunit composition of photosystem I and identification of center X as a [4Fe-4S] iron-sulfur cluster

A photosystem I (PS-I) preparation from barley (Hordeum vulgare L.) containing the reaction center protein P700-chlorophyll a-protein 1 (CP1) and smaller polypeptides with apparent molecular masses of 18, 16, 14, 9.5, 9, 4, and 1.5 kDa has been analyzed with respect to subunit stoichiometry. CP1 contains two homologous subunits with approximate masses of 82 kDa. CP1 and the smaller polypeptides were isolated, and the amino acid composition of each component and of the PS-I preparation was determined. Based on the amino acid composition data and the determined ability of each isolated polypeptide to bind Coomassie Brilliant Blue, the PS-I complex is shown to contain 1 mol of each of the homologous 82-kDa polypeptides as well as 1 mol of the 18-, 16-, 9.5-, and 9-kDa polypeptides for each mol of P700. The total polypeptide mass of the PS-I complex is 209 kDa excluding tryptophan and approximately 220 kDa including tryptophan. The two 82-kDa subunits present/P700 provide cysteine residues for binding only one Fe-S center. In conjunction with the earlier reported binding of four iron and four acid-labile sulfides to CP1/P700 (H?j, P. B., Svendsen, I., Scheller, H. V., and M?ller, B. L. (1987) J. Biol. Chem. 262, 12676-12684), this demonstrates the center X is a [4Fe-4S] cluster and eliminates the possibility of center X being composed of two [2Fe-2S] clusters.     

Resonance Raman studies of the [4Fe-4S] to [2Fe-2S] cluster conversion in the iron protein of nitrogenase

Resonance Raman spectroscopy has been used to investigate the Fe-S stretching modes of the [4Fe-4S]2+ cluster in the oxidized iron protein of Clostridium pasteurianum nitrogenase. The results are consistent with a cubane [4Fe-4S] cluster having effective Td symmetry with cysteinyl coordination for each iron. In accord with previous optical and EPR studies [(1984) Biochemistry 23, 2118-2122], treatment with the iron chelator alpha, alpha'-dipyridyl in the presence of MgATP is shown to effect cluster conversion to a [2Fe-2S]2+ cluster. Resonance Raman data also indicate that partial conversion to a [2Fe-2S]2+ cluster is induced by thionine-oxidation in the presence of MgATP in the absence of an iron chelator. This result suggests new explanations for the dramatic change in the CD spectrum that accompanies MgATP-binding to the oxidized Fe protein and the anomalous resonance Raman spectra of thionine-oxidized Clostridium pasteurianum bidirectional hydrogenase.     

Identification of variant molecules of Bacillus thermoproteolyticus ferredoxin: crystal structure reveals bound coenzyme A and an unexpected [3Fe-4S] cluster associated with a canonical [4Fe-4S] ligand motif

During the purification of recombinant Bacillus thermoproteolyticus ferredoxin (BtFd) from Escherichia coli, we have noted that some Fe-S proteins were produced in relatively small amounts compared to the originally identified BtFd carrying a [4Fe-4S] cluster. These variants could be purified into three Fe-S protein components (designated as V-I, V-II, and V-III) by standard chromatography procedures. UV-vis and EPR spectroscopic analyses indicated that each of these variants accommodates a [3Fe-4S] cluster. From mass spectrometric and protein sequence analyses together with native and SDS gel electrophoresis, we established that V-I and V-II contain the polypeptide of BtFd associated with acyl carrier protein (ACP) and with coenzyme A (CoA), respectively, and that V-III is a BtFd dimer linked by a disulfide bond. The crystal structure of the BtFd-CoA complex (V-II) determined at 1.6 A resolution revealed that each of the four complexes in the crystallographic asymmetric unit possesses a [3Fe-4S] cluster that is coordinated by Cys(11), Cys(17), and Cys(61). The polypeptide chain of each complex is superimposable onto that of the original [4Fe-4S] BtFd except for the segment containing Cys(14), the fourth ligand to the [4Fe-4S] cluster of BtFd. In the variant molecules, the side chain of Cys(14) is rotated away to the molecular surface, forming a disulfide bond with the terminal sulfhydryl group of CoA. This covalent modification may have occurred in vivo, thereby preventing the assembly of the [4Fe-4S] cluster as observed previously for Desulfovibrio gigas ferredoxin. Possibilities concerning how the variant molecules are formed in the cell are discussed.     

3Fe-4S] to [4Fe-4S] cluster conversion in Escherichia coli fumarate reductase by site-directed mutagenesis.

Site-directed mutants of Escherichia coli fumarate reductase in which FrdB Cys204, Cys210, and Cys214 were individually replaced by Ser and in which Val207 was replaced by Cys were constructed and overexpressed in a strain of E. coli lacking a wild-type copy of fumarate reductase and succinate dehydrogenase. The consequences of these mutations on bacterial growth, enzymatic activity, and the EPR properties of the constituent iron-sulfur clusters were investigated. The FrdB Cys204Ser, Cys210Ser, and Cys214Ser mutations result in enzymes with negligible activity that have dissociated from the membrane and consequently are incapable of supporting cell growth under conditions requiring a functional fumarate reductase. EPR studies indicate that these effects are associated with loss of both the [3Fe-4S] and [4Fe-4S] clusters, centers 3 and 2, respectively. In contrast, the FrdB Val207Cys mutation results in a functional membrane-bound enzyme that is able to support growth under anaerobic and aerobic conditions. However, EPR studies indicate that the indigenous [3Fe-4S]+,0 cluster (Em = -70 mV), center 3, has been replaced by a much lower potential [4Fe-4S]2+,+ cluster (Em = -350 mV), indicating that the primary sequence of the polypeptide determines the type of clusters assembled. The results of these studies afford new insights into the role of centers 2 and 3 in mediating electron transfer from menaquinol, the residues that ligate these clusters, and the intercluster magnetic interactions in the wild-type enzyme.     

Conversion of the central [4Fe-4S] cluster into a [3Fe-4S] cluster leads to reduced hydrogen-uptake activity of the F420-reducing hydrogenase of Methanococcus voltae.

As in many other hydrogenases, the small subunit of the F420-reducing hydrogenase of Methanococcus voltae contains three iron-sulfur clusters. The arrangement of the three [4Fe-4S] clusters corresponds to the arrangement of [Fe-S] clusters in the [NiFeSe] hydrogenase of Desulfomicrobium baculatum. Many other hydrogenases contain two [4Fe-4S] clusters and one [3Fe-4S] cluster with a relatively high redox potential, which is located in the central position between a proximal and a distal [4Fe-4S] cluster. We have investigated the role of the central [4Fe-4S] cluster in M. voltae with regard to its effect on the enzyme activity and its spectroscopic properties. Using site-directed mutagenesis, we constructed a strain in which one cysteine ligand of the central [4Fe-4S] cluster was replaced by proline. The mutant protein was purified, and the [4Fe-4S] to [3Fe-4S] cluster conversion was confirmed by EPR spectroscopy. The conversion resulted in an increase in the redox potential of the [3Fe-4S] cluster by about 400 mV. The [NiFe] active site was not affected significantly by the mutation as assessed by the unchanged Ni EPR spectrum. The specific activity of the mutated enzyme did not show any significant differences with the artificial electron acceptor benzyl viologen, but its specific activity with the natural electron acceptor F420 decreased tenfold.     

Escherichia coli L-serine deaminase requires a [4Fe-4S] cluster in catalysis

L-Serine deaminases catalyze the deamination of L-serine, producing pyruvate and ammonia. Two families of these proteins have been described and are delineated by the cofactor that each employs in catalysis. These are the pyridoxal 5'-phosphate-dependent deaminases and the deaminases that are activated in vitro by iron and dithiothreitol. In contrast to the enzymes that employ pyridoxal 5'-phosphate, detailed physical and mechanistic characterization of the iron-dependent deaminases is limited, primarily because of their extreme instability. We report here the characterization of L-serine deaminase from Escherichia coli, which is the product of the sdaA gene. When purified anaerobically, the isolated protein contains 1.86 +/- 0.46 eq of iron and 0.670 +/- 0.019 eq of sulfide per polypeptide and displays a UV-visible spectrum that is consistent with a [4Fe-4S] cluster. Reconstitution of the protein with iron and sulfide generates considerably more of the cluster, and treatment of the reconstituted protein with dithionite gives rise to an axial EPR spectrum, displaying g axially = 2.03 and g radially = 1.93. M?ssbauer spectra of the (57)Fe-reconstituted protein reveal that the majority of the iron is in the form of [4Fe-4S](2+) clusters, as evidenced by the typical M?ssbauer parameters-isomer shift, delta = 0.47 mm/s, quadrupole splitting of Delta E(Q) = 1.14 mm/s, and a diamagnetic (S = 0) ground state. Treatment of the dithionite-reduced protein with L-serine results in a slight broadening of the feature at g = 2.03 in the EPR spectrum of the protein, and a dramatic loss in signal intensity, suggesting that the amino acid interacts directly with the cluster.     

Iron-sulfur cluster interconversions in biotin synthase: dissociation and reassociation of iron during conversion of [2Fe-2S] to [4Fe-4S] clusters

Biotin synthase catalyzes the insertion of a sulfur atom into the saturated C6 and C9 carbons of dethiobiotin. This reaction has long been presumed to occur through radical chemistry, and recent experimental results suggest that biotin synthase belongs to a family of enzymes that contain an iron-sulfur cluster and reductively cleave S-adenosylmethionine, forming an enzyme or substrate radical, 5'-deoxyadenosine, and methionine. Biotin synthase (BioB) is aerobically purified as a dimer of 38 kDa monomers that contains two [2Fe-2S](2+) clusters per dimer. Maximal in vitro biotin synthesis requires incubation of BioB with dethiobiotin, AdoMet, reductants, exogenous iron, and crude bacterial protein extracts. It has previously been shown that reduction of BioB with dithionite in 60% ethylene glycol produces one [4Fe-4S](2+/1+) cluster per dimer. In the present work, we use UV/visible and electron paramagnetic resonance spectroscopy to show that [2Fe-2S] to [4Fe-4S] cluster conversion occurs through rapid dissociation of iron from the protein followed by rate-limiting reassociation. While in 60% ethylene glycol the product of dithionite reduction is one [4Fe-4S](2+) cluster per dimer, the product in water is one [4Fe-4S](1+) cluster per dimer. Further, incubation with excess iron, sulfide, and dithiothreitol produces protein that contains two [4Fe-4S](2+) clusters per dimer; subsequent reduction with dithionite produces two [4Fe-4S](1+) clusters per BioB dimer. BioB that contains two [4Fe-4S](2+/1+) clusters per dimer is rapidly and reversibly reduced and oxidized, suggesting that this is the redox-active form of the iron-sulfur cluster in the anaerobic enzyme.     

Evidence for the formation of a linear [3Fe-4S] cluster in partially unfolded aconitase

Beef heart aconitase, as isolated under aerobic conditions, is inactive and contains a [3Fe-4S]1+ cluster. On incubation at pH greater than 9.5 (or treatment with 4-8 M urea) the color of the protein changes from brown to purple. This purple form is stable and can be converted back in good yield to the active [4Fe-4S]2+ form by reduction in the presence of iron. Active aconitase is converted to the purple form at alkaline pH only after oxidative inactivation. The Fe/S2- ratio of purple aconitase is 0.8, indicating the presence of [3Fe-4S] clusters. The number of SH groups readily reacting with 5,5'-dithiobis(2-nitrobenzoic acid) is increased from approximately 1 in the enzyme as isolated to 7-8 in the purple form, indicating a partial unfolding of the protein. On conversion of inactive aconitase to the purple form, the EPR signal at g = 2.01 (S = 1/2) is replaced by signals at g = 4.3 and 9.6 (S = 5/2). M?ssbauer spectroscopy shows that purple aconitase has high-spin ferric ions, each residing in a tetrahedral environment of sulfur atoms. The three iron sites are exchange-coupled to yield a ground state with S = 5/2. Analysis of the data within a spin coupling model shows that J13 congruent to J23 and 2 J12 less than J13, where the Jik describe the antiferromagnetic (J greater than 0) exchange interactions among the three iron pairs. Comparison of our data with those reported for synthetic Fe-S clusters (Hagen, K. S., Watson, A. D., and Holm, R. H., (1983) J. Am. Chem. Soc. 105, 3905-3913) shows that purple aconitase contains a linear [3Fe-4S]1+ cluster, a structural isomer of the S = 1/2 cluster of inactive aconitase. Our studies also show that protein-bound [2Fe-2S] clusters can be generated under conditions where partial unfolding of the protein occurs.     

Evidence that centre 2 in Escherichia coli fumarate reductase is a [4Fe-4S]cluster

Redox titrations of the iron-sulphur clusters in fumarate reductase purified from Escherichia coli, monitored by ESR spectroscopy, identified three redox events, similar to those observed in other fumarate reductases and succinate dehydrogenases: Centre 1, a [2Fe-2S] cluster, at g = 2.03, 1.93, appeared on reduction with Em = -20 mV. Centre 3, probably a [3Fe-xS] cluster, at g = 2.02 appeared in the oxidized state with Em = -70 mV. Centre 2 has been observed as an increase in the electron-spin relaxation of Centre 1. It titrates as an n = 1 species with Em = -320 mV, but in our hands did not appear to contribute significant intensity to the g = 2.03, 1.93 signal. It therefore appears to be an additional centre which undergoes spin-spin interaction with Centre 1. The reduction of Centre 2 coincided with the appearance of an extremely broad ESR spectrum, observed at temperatures below 20 K, with features at g = 2.17, 1.9, 1.68. The broad signal was observed in both soluble and membrane-bound preparations. Its midpoint potential was -320 mV. Its integrated intensity was approximately equal to that of Centre 1, if its broad outer wings were taken into account. Consideration of the ESR properties of this signal, together with the amino acid sequence of the frdB subunit of the enzyme, indicates that Centre 2 is a [4Fe-4S] cluster which, in its reduced state, enhances the spin relaxation of the [2Fe-2S] Centre 1.     

HscA and HscB stimulate [2Fe-2S] cluster transfer from IscU to apoferredoxin in an ATP-dependent reaction

The role of the Azotobacter vinelandii HscA/HscB cochaperone system in ISC-mediated iron-sulfur cluster biogenesis has been investigated in vitro by using CD and EPR spectrometry to monitor the effect of HscA, HscB, MgATP, and MgADP on the time course of cluster transfer from [2Fe-2S]IscU to apo-Isc ferredoxin. CD spectra indicate that both HscB and HscA interact with [2Fe-2S]IscU and the rate of cluster transfer was stimulated more than 20-fold in the presence stoichiometric HscA and HscB and excess MgATP. No stimulation was observed in the absence of either HscB or MgATP, and cluster transfer was found to be an ATP-dependent reaction based on concomitant phosphate production and the enhanced rates of cluster transfer in the presence of KCl which is known to stimulate HscA ATPase activity. The results demonstrate a role of the ISC HscA/HscB cochaperone system in facilitating efficient [2Fe-2S] cluster transfer from the IscU scaffold protein to acceptor proteins and that [2Fe-2S] cluster transfer from IscU is an ATP-dependent process. The data are consistent with the proposed regulation of the HscA ATPase cycle by HscB and IscU [Silberg, J. J., Tapley, T. L., Hoff, K. G., and Vickery, L. E. (2004) J. Biol. Chem. 279, 53924-53931], and mechanistic proposals for coupling of the HscA ATPase cycle with cluster transfer from [2Fe-2S]IscU to apo-IscFdx are discussed.     

Crystal structure of the 2[4Fe-4S] ferredoxin from Chromatium vinosum: evolutionary and mechanistic inferences for [3/4Fe-4S] ferredoxins.

The crystal structure of the 2[4Fe-4S] ferredoxin from Chromatium vinosum has been solved by molecular replacement using data recorded with synchrotron radiation. The crystals were hexagonal prisms that showed a strong tendency to develop into long tubes. The hexagonal prisms diffracted to 2.1 A resolution at best, and a structural model for C. vinosum ferredoxin has been built with a final R of 19.2%. The N-terminal domain coordinates the two [4Fe-4S] clusters in a fold that is almost identical to that of other known ferredoxins. However, the structure has two unique features. One is a six-residue insertion between two ligands of one cluster forming a two-turn external loop; this short loop changes the conformation of the Cys 40 ligand compared to other ferredoxins and hampers the building of one NH...S H-bond to one of the inorganic sulfurs. The other remarkable structural element is a 3.5-turn alpha-helix at the C-terminus that covers one side of the same cluster and is linked to the cluster-binding domain by a six-residue external chain segment. The charge distribution is highly asymmetric over the molecule. The structure of C. vinosum ferredoxin strongly suggests divergent evolution for bacterial [3/4Fe-4S] ferredoxins from a common ancestral cluster-binding core. The unexpected slow intramolecular electron transfer rate between the clusters in C. vinosum ferredoxin, compared to other similar proteins, may be attributed to the unusual electronic properties of one of the clusters arising from localized changes in its vicinity rather than to a global structural rearrangement.     

Structure of a xanthine oxidase-related 4-hydroxybenzoyl-CoA reductase with an additional [4Fe-4S] cluster and an inverted electron flow

The Mo-flavo-Fe/S-dependent heterohexameric protein complex 4-hydroxybenzoyl-CoA reductase (4-HBCR, dehydroxylating) is a central enzyme of the anaerobic degradation of phenolic compounds and belongs to the xanthine oxidase (XO) family of molybdenum enzymes. Its X-ray structure was established at 1.6 A resolution. The most pronounced difference between 4-HBCR and other structurally characterized members of the XO family is the insertion of 40 amino acids within the beta subunit, which carries an additional [4Fe-4S] cluster at a distance of 16.5 A to the isoalloxazine ring of FAD. The architecture of 4-HBCR and concomitantly performed electron transfer rate calculations suggest an inverted electron transfer chain from the donor ferredoxin via the [4Fe-4S] cluster to the Mo over a distance of 55 A. The binding site of 4-hydroxybenzoyl-CoA is located in an 18 A long channel lined up by several aromatic side chains around the aromatic moiety, which are proposed to shield and stabilize the postulated radical intermediates during catalysis.     

Dihydroxy acid dehydratase from spinach contains a [2Fe-2S] cluster

Dihydroxy acid dehydratase, the third enzyme in the branched-chain amino acid biosynthetic pathway, has been purified to homogeneity (5000-fold) from spinach leaves. The molecular weights of dihydroxy acid dehydratase as determined by sodium dodecyl sulfate and native gel electrophoresis are 63,000 and 110,000, respectively, suggesting the native enzyme is a dimer. 2 moles of iron were found per mol of protein monomer. Chemical analyses of iron and labile sulfide gave an Fe/S2- ratio of 0.95. The EPR spectrum of dithionite-reduced enzyme (gavg = 1.91) is similar to spectra characteristic of Rieske Fe-S proteins and has a spin concentration of 1 spin/1.9 irons. These results strongly suggest that dihydroxy acid dehydratase contains a [2Fe-2S] cluster, a novel finding for enzymes of the hydrolyase class. In contrast to the Rieske Fe-S proteins, the redox potential of the Fe-S cluster is quite low (-470 mV). Upon addition of substrate, the EPR signal of the reduced enzyme changes to one typical of 2Fe ferredoxins (gavg = 1.95), and the visible absorption spectrum of the native enzyme shows substantial changes between 400 and 600 nm. Reduction of the Fe-S cluster decreases the enzyme activity by 6-fold under Vmax conditions. These results suggest the direct involvement of the [2Fe-2S] cluster of dihydroxy acid dehydratase in catalysis. Similar conclusions have been reached for the catalytic involvement of the [4Fe-4S] cluster of the hydrolyase aconitase (Emptage, M. H., Kent, T. A., Kennedy, M. C., Beinert, H., and Münck, E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4674-4678).     

Characterization of cluster N5 as a fast-relaxing [4Fe-4S] cluster in the Nqo3 subunit of the proton-translocating NADH-ubiquinone oxidoreductase from Paracoccus denitrificans

The NADH-quinone oxidoreductase from Paracoccus denitrificans consists of 14 subunits (Nqo1-14) and contains one FMN and eight iron-sulfur clusters. The Nqo3 subunit possesses fully conserved 11 Cys and 1 His in its N-terminal region and is considered to harbor three iron-sulfur clusters; however, only one binuclear (N1b) and one tetranuclear (N4) were previously identified. In this study, the Nqo3 subunit containing 1x[2Fe-2S] and 2x[4Fe-4S] clusters was expressed in Escherichia coli. The second [4Fe-4S](1+) cluster is detected by EPR spectroscopy below 6 K, exhibiting very fast spin relaxation. The resolved EPR spectrum of this cluster is broad and nearly axial. The subunit exhibits an absorption-type EPR signal around g approximately 5 region below 6 K, most likely arising from an S = 3/2 ground state of the fast-relaxing [4Fe-4S](1+) species. The substitution of the conserved His(106) with Cys specifically affected the fast-relaxing [4Fe-4S](1+) cluster, suggesting that this cluster is coordinated by His(106). In the cholate-treated NDH-1-enriched P. denitrificans membranes, we observed EPR signals arising from a [4Fe-4S] cluster below 6 K, exhibiting properties similar to those of cluster N5 detected in other complex I/NDH-1 and of the fast-relaxing [4Fe-4S](1+) cluster in the expressed Nqo3 subunit. Hence, we propose that the His-coordinated [4Fe-4S] cluster corresponds to cluster N5.     

Formation of a linear [3Fe-4S] cluster in a seven-iron ferredoxin triggered by polypeptide unfolding

Cubic iron-sulfur ([Fe-S]) clusters are common inorganic cofactors in proteins. The presence of a linear [3Fe-4S] cluster in a protein was first observed in beef-heart aconitase at high pH, where the protein structure was perturbed. Not long ago, the same linear cluster was discovered upon unfolding of a thermophilic di-cluster seven-iron ferredoxin, suggesting a more general relevance for this type of linear clusters in Nature. Since structure-induced cluster rearrangements may be important regulatory, on-going processes in living systems, we decided to further characterize the formation of the linear iron-sulfur cluster observed upon ferredoxin unfolding. Here we present a kinetic investigation of parameters that affect the linear-cluster formation and disassembly in the Sulfolobus acidocaldarius seven-iron ferredoxin. We find the linear cluster to be an intermediate on the protein-mediated cluster-degradation pathway under a wide range of pH and denaturant conditions. The linear species forms in parallel with secondary-structure disappearance. In contrast, the disassembly rate constant for the linear cluster is independent of denaturant concentration but depends strongly on solution pH. At high pH, the disassembly rate is slower and the linear iron-sulfur species has a longer lifetime, than at low pH.     

Identification of [2Fe-2S] clusters in microbial ferrochelatases

The terminal enzyme of heme biosynthesis, ferrochelatase (EC 4.99.1.1), catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme. Prior to the present work, [2Fe-2S] clusters have been identified and characterized in animal ferrochelatases but not in plant or prokaryotic ferrochelatases. Herein we present evidence that ferrochelatases from the bacteria Caulobacter crescentus and Mycobacterium tuberculosis possess [2Fe-2S] clusters. The enzyme from C. crescentus is a homodimeric, membrane-associated protein while the enzyme from M. tuberculosis is monomeric and soluble. The clusters of the C. crescentus and M. tuberculosis ferrochelatases are ligated by four cysteines but possess ligand spacings that are unlike those of any previously characterized [2Fe-2S] cluster-containing protein, including the ferrochelatase of the yeast Schizosaccharomyces pombe. Thus, the microbial ferrochelatases represent a new group of [2Fe-2S] cluster-containing proteins.