The novel structure and chemistry of iron-sulfur clusters in the adenosylmethionine-dependent radical enzyme biotin synthase

Biotin synthase is an adenosylmethionine-dependent radical enzyme that catalyzes the substitution of sulfur for hydrogen at the saturated C6 and C9 positions in dethiobiotin. The structure of the biotin synthase monomer is an (alpha/beta)(8) barrel that contains one [4Fe-4S](2+) cluster and one [2Fe-2S](2+) cluster that encapsulate the substrates AdoMet and dethiobiotin. The air-sensitive [4Fe-4S](2+) cluster and the reductant-sensitive [2Fe-2S](2+) cluster have unique coordination environments that include close proximity to AdoMet and DTB, respectively. The relative positioning of these components, as well as several conserved protein residues, suggests at least two potential catalytic mechanisms that incorporate sulfur from either the [2Fe-2S](2+) cluster or a cysteine persulfide into the biotin thiophane ring. This review summarizes an accumulating consensus regarding the physical and spectroscopic properties of each FeS cluster, and discusses possible roles for the [4Fe-4S](2+) cluster in radical generation and the [2Fe-2S](2+) cluster in sulfur incorporation.     

Biotin synthase contains two distinct iron-sulfur cluster binding sites: chemical and spectroelectrochemical analysis of iron-sulfur cluster interconversions.

Biotin synthase is an iron-sulfur protein that utilizes AdoMet to catalyze the presumed radical-mediated insertion of a sulfur atom between the saturated C6 and C9 carbons of dethiobiotin. Biotin synthase (BioB) is aerobically purified as a dimer that contains [2Fe-2S](2+) clusters and is inactive in the absence of additional iron and reductants, and anaerobic reduction of BioB with sodium dithionite results in conversion to enzyme containing [4Fe-4S](2+) and/or [4Fe-4S](+) clusters. To establish the predominant cluster forms present in biotin synthase in anaerobic assays, and by inference in Escherichia coli, we have accurately determined the extinction coefficient and cluster content of the enzyme under oxidized and reduced conditions and have examined the equilibrium reduction potentials at which cluster reductions and conversions occur as monitored by UV/visible and EPR spectroscopy. In contrast to previous reports, we find that aerobically purified BioB contains ca. 1.2-1.5 [2Fe-2S](2+) clusters per monomer with epsilon(452) = 8400 M(-)(1) cm(-)(1) per monomer. Upon reduction, the [2Fe-2S](2+) clusters are converted to [4Fe-4S] clusters with two widely separate reduction potentials of -140 and -430 mV. BioB reconstituted with excess iron and sulfide in 60% ethylene glycol was found to contain two [4Fe-4S](2+) clusters per monomer with epsilon(400) = 30 000 M(-)(1) cm(-)(1) per monomer and is reduced with lower midpoint potentials of -440 and -505 mV, respectively. Finally, as predicted by the measured redox potentials, enzyme incubated under typical anaerobic assay conditions is repurified containing one [2Fe-2S](2+) cluster and one [4Fe-4S](2+) cluster per monomer. These results indicate that the dominant stable cluster state for biotin synthase is a dimer containing two [2Fe-2S](2+) and two [4Fe-4S](2+) clusters.     

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.     

Spectroscopic changes during a single turnover of biotin synthase: destruction of a [2Fe-2S] cluster accompanies sulfur insertion.

Biotin synthase catalyzes the insertion of a sulfur atom between the saturated C6 and C9 carbons of dethiobiotin. Catalysis requires AdoMet and flavodoxin and generates 5'-deoxyadenosine and methionine, suggesting that biotin synthase is an AdoMet-dependent radical enzyme. Biotin synthase (BioB) is aerobically purified as a dimer of 38.4 kDa monomers that contains 1-1.5 [2Fe-2S](2+) clusters per monomer and can be reconstituted with exogenous iron, sulfide, and reductants to contain up to two [4Fe-4S] clusters per monomer. The iron-sulfur clusters may play a dual role in biotin synthase: a reduced iron-sulfur cluster is probably involved in radical generation by mediating the reductive cleavage of AdoMet, while recent in vitro labeling studies suggest that an iron-sulfur cluster also serves as the immediate source of sulfur for the biotin thioether ring. Consistent with this dual role for iron-sulfur clusters in biotin synthase, we have found that the protein is stable, containing one [2Fe-2S](2+) cluster and one [4Fe-4S](2+) cluster per monomer. In the present study, we demonstrate that this mixed cluster state is essential for optimal activity. We follow changes in the Fe and S content and UV/visible and EPR spectra of the enzyme during a single turnover and conclude that during catalysis the [4Fe-4S](2+) cluster is preserved while the [2Fe-2S](2+) cluster is destroyed. We propose a mechanism for incorporation of sulfur into dethiobiotin in which a sulfur atom is oxidatively extracted from the [2Fe-2S](2+) 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.     

Iron-sulfur clusters of biotin synthase in vivo: a Mössbauer study

Biotin synthase, the enzyme that catalyzes the last step of the biosynthesis of biotin, contains only [2Fe-2S](2+) clusters when isolated under aerobic conditions. Previous results showed that reconstitution with an excess of FeCl(3) and Na(2)S under reducing and anaerobic conditions leads to either [4Fe-4S](2+), [4Fe-4S](+), or a mixture of [4Fe-4S](2+) and [2Fe-2S](2+) clusters. To determine whether any of these possibilities or other different cluster configuration could correspond to the physiological in vivo state, we have used (57)Fe M?ssbauer spectroscopy to investigate the clusters of biotin synthase in whole cells. The results show that, in aerobically grown cells, biotin synthase contains a mixture of [4Fe-4S](2+) and [2Fe-2S](2+) clusters. A mixed [4Fe-4S](2+):[2Fe-2S](2+) cluster form has already been observed under certain in vitro conditions, and it has been proposed that both clusters might each play a significant role in the mechanism of biotin synthase. Their presence in vivo is now another argument in favor of this mixed cluster form.     

Reductive cleavage of S-adenosylmethionine by biotin synthase from Escherichia coli

Biotin synthase (BioB) catalyzes the insertion of a sulfur atom between the C6 and C9 carbons of dethiobiotin. Reconstituted BioB from Escherichia coli contains a [4Fe-4S](2+/1+) cluster thought to be involved in the reduction and cleavage of S-adenosylmethionine (AdoMet), generating methionine and the reactive 5'-deoxyadenosyl radical responsible for dethiobiotin H-abstraction. Using EPR and M?ssbauer spectroscopy as well as methionine quantitation we demonstrate that the reduced S = 1/2 [4Fe-4S](1+) cluster is indeed capable of injecting one electron into AdoMet, generating one equivalent of both methionine and S = 0 [4Fe-4S](2+) cluster. Dethiobiotin is not required for the reaction. Using site-directed mutagenesis we show also that, among the eight cysteines of BioB, only three (Cys-53, Cys-57, Cys-60) are essential for AdoMet reductive cleavage, suggesting that these cysteines are involved in chelation of the [4Fe-4S](2+/1+) cluster.     

Structural organization of essential iron-sulfur clusters in the evolutionarily highly conserved ATP-binding cassette protein ABCE1

The ABC protein ABCE1, formerly named RNase L inhibitor RLI1, is one of the most conserved proteins in evolution and is expressed in all organisms except eubacteria. Because of its fundamental role in translation initiation and/or ribosome biosynthesis, ABCE1 is essential for life. Its molecular mechanism has, however, not been elucidated. In addition to two ABC ATPase domains, ABCE1 contains a unique N-terminal region with eight conserved cysteines, predicted to coordinate iron-sulfur clusters. Here we present detailed information on the type and on the structural organization of the Fe-S clusters in ABCE1. Based on biophysical, biochemical, and yeast genetic analyses, ABCE1 harbors two essential diamagnetic [4Fe-4S](2+) clusters with different electronic environments, one ferredoxin-like (CPX(n)CX(2)CX(2)C; Cys at positions 4-7) and one unique ABCE1-type cluster (CXPX(2)CX(3)CX(n)CP; Cys at positions 1, 2, 3, and 8). Strikingly, only seven of the eight conserved cysteines coordinating the Fe-S clusters are essential for cell viability. Mutagenesis of the cysteine at position 6 yielded a functional ABCE1 with the ferredoxin-like Fe-S cluster in a paramagnetic [3Fe-4S](+) state. Notably, a lethal mutation of the cysteine at position 4 can be rescued by ligand swapping with an adjacent, extra cysteine conserved among all eukaryotes.     

Biotin synthase mechanism: mutagenesis of the YNHNLD conserved motif

Biotin synthase, a member of the "radical SAM" family, catalyzes the final step of the biotin biosynthetic pathway, namely, the insertion of a sulfur atom into dethiobiotin (DTB). The active form of the enzyme contains two iron-sulfur clusters, a [4Fe-4S](2+) cluster liganded by Cys-53, Cys-57, and Cys-60 and the S-adenosylmethionine (AdoMet or SAM) cosubstrate and a [2Fe-2S](2+) cluster liganded by Cys-97, Cys-128, Cys-188, and Arg-260. Single-point mutation of each of these six conserved cysteines produced inactive variants. In this work, mutants of other highly conserved residues from the Y(150)NHNLD motif are described. They have properties similar to those of the wild-type enzyme with respect to their cluster content and characteristics. For all of them, the as-isolated form, which contains an air-stable [2Fe-2S](2+) center, can additionally accommodate an air-sensitive [4Fe-4S](2+) center which is generated by incubation under anaerobic conditions with Fe(2+) and S(2-). Their spectroscopic properties are similar to those of the wild type. However, they are inactive, except the mutant H152A that exhibits a weak activity. We show that the mutants, inactive in producing biotin, are also unable to cleave AdoMet and to produce the deoxyadenosyl radical (AdoCH(2)(*)). In the case of H152A, a value of 5.5 +/- 0.4 is found for the 5'-deoxyadenosine (AdoCH(3)):biotin ratio, much higher than the value of 2.8 +/- 0.3 usually observed with the wild type. This reveals a greater contribution of the abortive process in which the AdoCH(2)(*) radical is quenched by hydrogen atoms from the protein or from some components of the system. Thus, in this case, the coupling between the production of AdoCH(2)(*) and its reaction with the hydrogen at C-6 and C-9 of DTB is less efficient than that in the wild type, probably because of geometry's perturbation within the active site.     

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.     

Role of the [2Fe-2S] cluster in recombinant Escherichia coli biotin synthase

Biotin synthase (BioB) converts dethiobiotin into biotin by inserting a sulfur atom between C6 and C9 of dethiobiotin in an S-adenosylmethionine (SAM)-dependent reaction. The as-purified recombinant BioB from Escherichia coli is a homodimeric molecule containing one [2Fe-2S](2+) cluster per monomer. It is inactive in vitro without the addition of exogenous Fe. Anaerobic reconstitution of the as-purified [2Fe-2S]-containing BioB with Fe(2+) and S(2)(-) produces a form of BioB that contains approximately one [2Fe-2S](2+) and one [4Fe-4S](2+) cluster per monomer ([2Fe-2S]/[4Fe-4S] BioB). In the absence of added Fe, the [2Fe-2S]/[4Fe-4S] BioB is active and can produce up to approximately 0.7 equiv of biotin per monomer. To better define the roles of the Fe-S clusters in the BioB reaction, M?ssbauer and electron paramagnetic resonance (EPR) spectroscopy have been used to monitor the states of the Fe-S clusters during the conversion of dethiobiotin to biotin. The results show that the [4Fe-4S](2+) cluster is stable during the reaction and present in the SAM-bound form, supporting the current consensus that the functional role of the [4Fe-4S] cluster is to bind SAM and facilitate the reductive cleavage of SAM to generate the catalytically essential 5'-deoxyadenosyl radical. The results also demonstrate that approximately (2)/(3) of the [2Fe-2S] clusters are degraded by the end of the turnover experiment (24 h at 25 degrees C). A transient species with spectroscopic properties consistent with a [2Fe-2S](+) cluster is observed during turnover, suggesting that the degradation of the [2Fe-2S](2+) cluster is initiated by reduction of the cluster. This observed degradation of the [2Fe-2S] cluster during biotin formation is consistent with the proposed sacrificial S-donating function of the [2Fe-2S] cluster put forth by Jarrett and co-workers (Ugulava et al. (2001) Biochemistry 40, 8352-8358). Interestingly, degradation of the [2Fe-2S](2+) cluster was found not to parallel biotin formation. The initial decay rate of the [2Fe-2S](2+) cluster is about 1 order of magnitude faster than the initial formation rate of biotin, indicating that if the [2Fe-2S] cluster is the immediate S donor for biotin synthesis, insertion of S into dethiobiotin would not be the rate-limiting step. Alternatively, the [2Fe-2S] cluster may not be the immediate S donor. Instead, degradation of the [2Fe-2S] cluster may generate a protein-bound polysulfide or persulfide that serves as the immediate S donor for biotin production.     

Reduction of the [2Fe-2S] cluster accompanies formation of the intermediate 9-mercaptodethiobiotin in Escherichia coli biotin synthase

Biotin synthase catalyzes the conversion of dethiobiotin (DTB) to biotin through the oxidative addition of sulfur between two saturated carbon atoms, generating a thiophane ring fused to the existing ureido ring. Biotin synthase is a member of the radical SAM superfamily, composed of enzymes that reductively cleave S-adenosyl-l-methionine (SAM or AdoMet) to generate a 5'-deoxyadenosyl radical that can abstract unactivated hydrogen atoms from a variety of organic substrates. In biotin synthase, abstraction of a hydrogen atom from the C9 methyl group of DTB would result in formation of a dethiobiotinyl methylene carbon radical, which is then quenched by a sulfur atom to form a new carbon-sulfur bond in the intermediate 9-mercaptodethiobiotin (MDTB). We have proposed that this sulfur atom is the μ-sulfide of a [2Fe-2S](2+) cluster found near DTB in the enzyme active site. In the present work, we show that formation of MDTB is accompanied by stoichiometric generation of a paramagnetic FeS cluster. The electron paramagnetic resonance (EPR) spectrum is modeled as a 2:1 mixture of components attributable to different forms of a [2Fe-2S](+) cluster, possibly distinguished by slightly different coordination environments. Mutation of Arg260, one of the ligands to the [2Fe-2S] cluster, causes a distinctive change in the EPR spectrum. Furthermore, magnetic coupling of the unpaired electron with (14)N from Arg260, detectable by electron spin envelope modulation (ESEEM) spectroscopy, is observed in WT enzyme but not in the Arg260Met mutant enzyme. Both results indicate that the paramagnetic FeS cluster formed during catalytic turnover is a [2Fe-2S](+) cluster, consistent with a mechanism in which the [2Fe-2S](2+) cluster simultaneously provides and oxidizes sulfide during carbon-sulfur bond formation.     

Escherichia coli biotin synthase produces selenobiotin. Further evidence of the involvement of the [2Fe-2S]2+ cluster in the sulfur insertion step

Biotin synthase, a member of the "radical SAM" family, catalyzes the final step of the biotin biosynthetic pathway, namely, the insertion of a sulfur atom into dethiobiotin. The as-isolated enzyme contains a [2Fe-2S](2+) cluster, but the active enzyme requires an additional [4Fe-4S](2+) cluster, which is formed in the presence of Fe(NH(4))(2)(SO(4))(2) and Na(2)S in the in vitro assay. The role of the [4Fe-4S](2+) cluster is to mediate the electron transfer to SAM, while the [2Fe-2S](2+) cluster is involved in the sulfur insertion step. To investigate the selenium version of the reaction, we have depleted the enzyme of its iron and sulfur and reconstituted the resulting apoprotein with FeCl(3) and Na(2)Se to yield a [2Fe-2Se](2+) cluster. This enzyme was assayed in vitro with Na(2)Se in place of Na(2)S to enable the formation of a [4Fe-4Se](2+) cluster. Selenobiotin was produced, but the activity was lower than that of the as-isolated [2Fe-2S](2+) enzyme in the presence of Na(2)S. The [2Fe-2Se](2+) enzyme was additionally assayed with Na(2)S, to reconstitute a [4Fe-4S](2+) cluster, in case the latter was more efficient than a [4Fe-4Se](2+) cluster for the electron transfer. Indeed, the activity was improved, but in that case, a mixture of biotin and selenobiotin was produced. This was unexpected if one considers the [2Fe-2S](2+) center as the sulfur source (either as the ultimate donor or via another intermediate), unless some exchange of the chalcogenide has taken place in the cluster. This latter point was seen in the resonance Raman spectrum of the reacted enzyme which clearly indicated the presence of both the [2Fe-2Se](2+) and [2Fe-2S](2+) clusters. No exchange was observed in the absence of reaction. These observations bring supplementary proof that the [2Fe-2S](2+) cluster is implicated in the sulfur insertion step.     

Activation of class III ribonucleotide reductase from E. coli. The electron transfer from the iron-sulfur center to S-adenosylmethionine

The anaerobic ribonucleotide reductase (ARR) from E. coli is the prototype for enzymes that use the combination of S-adenosylmethionine (AdoMet) and an iron-sulfur center for generating catalytically essential free radicals. ARR is a homodimeric alpha2 protein which acquires a glycyl radical during anaerobic incubation with a [4Fe-4S]-containing activating enzyme (beta) and AdoMet under reducing conditions. Here we show that the EPR-active S = 1/2 reduced [4Fe-4S]+ cluster is competent for AdoMet reductive cleavage, yielding 1 equiv of methionine and almost 1 equiv of glycyl radical. These data support the proposal that the glycyl radical results from a one-electron oxidation of the reduced cluster by AdoMet. Reduced protein beta alone is also able to reduce AdoMet but only in the presence of DTT. However, in that case, 2 equiv of methionine per reduced cluster was formed. This unusual stoichiometry and combined EPR and M?ssbauer spectroscopic analysis are used to tentatively propose that AdoMet reductive cleavage proceeds by an alternative mechanism involving catalytically active [3Fe-4S] intermediate clusters.     

Structure of C42D Azotobacter vinelandii FdI. A Cys-X-X-Asp-X-X-Cys motif ligates an air-stable [4Fe-4S]2+/+ cluster

All naturally occurring ferredoxins that have Cys-X-X-Asp-X-X-Cys motifs contain [4Fe-4S](2+/+) clusters that can be easily and reversibly converted to [3Fe-4S](+/0) clusters. In contrast, ferredoxins with unmodified Cys-X-X-Cys-X-X-Cys motifs assemble [4Fe-4S](2+/+) clusters that cannot be easily interconverted with [3Fe-4S](+/0) clusters. In this study we changed the central cysteine of the Cys(39)-X-X-Cys(42)-X-X-Cys(45) of Azotobacter vinelandii FdI, which coordinates its [4Fe-4S](2+/+) cluster, into an aspartate. UV-visible, EPR, and CD spectroscopies, metal analysis, and x-ray crystallography show that, like native FdI, aerobically purified C42D FdI is a seven-iron protein retaining its [4Fe-4S](2+/+) cluster with monodentate aspartate ligation to one iron. Unlike known clusters of this type the reduced [4Fe-4S](+) cluster of C42D FdI exhibits only an S = 1/2 EPR with no higher spin signals detected. The cluster shows only a minor change in reduction potential relative to the native protein. All attempts to convert the cluster to a 3Fe cluster using conventional methods of oxygen or ferricyanide oxidation or thiol exchange were not successful. The cluster conversion was ultimately accomplished using a new electrochemical method. Hydrophobic and electrostatic interaction and the lack of Gly residues adjacent to the Asp ligand explain the remarkable stability of this cluster.     

NifS-mediated assembly of [4Fe-4S] clusters in the N- and C-terminal domains of the NifU scaffold protein

NifU is a homodimeric modular protein comprising N- and C-terminal domains and a central domain with a redox-active [2Fe-2S](2+,+) cluster. It plays a crucial role as a scaffold protein for the assembly of the Fe-S clusters required for the maturation of nif-specific Fe-S proteins. In this work, the time course and products of in vitro NifS-mediated iron-sulfur cluster assembly on full-length NifU and truncated forms involving only the N-terminal domain or the central and C-terminal domains have been investigated using UV-vis absorption and M?ssbauer spectroscopies, coupled with analytical studies. The results demonstrate sequential assembly of labile [2Fe-2S](2+) and [4Fe-4S](2+) clusters in the U-type N-terminal scaffolding domain and the assembly of [4Fe-4S](2+) clusters in the Nfu-type C-terminal scaffolding domain. Both scaffolding domains of NifU are shown to be competent for in vitro maturation of nitrogenase component proteins, as evidenced by rapid transfer of [4Fe-4S](2+) clusters preassembled on either the N- or C-terminal domains to the apo nitrogenase Fe protein. Mutagenesis studies indicate that a conserved aspartate (Asp37) plays a critical role in mediating cluster transfer. The assembly and transfer of clusters on NifU are compared with results reported for U- and Nfu-type scaffold proteins, and the need for two functional Fe-S cluster scaffolding domains on NifU is discussed.     

Chemical rescue of a site-modified ligand to a [4Fe-4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to Photosystem I

Chemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure-function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron-sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe-4S](1+,2+) cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe-4S](1+,2+) clusters termed F(A) and F(B). In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron-sulfur cluster F(B). Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and M?ssbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe-4S](1+,2+) clusters, despite the absence of one of the biological ligands. (19)F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the F(B) cluster. The finding that site-modified [4Fe-4S](1+,2+) clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand.     

The Radical SAM Superfamily

The radical S-adenosylmethionine (SAM) superfamily currently comprises more than 2800 proteins with the amino acid sequence motif CxxxCxxC unaccompanied by a fourth conserved cysteine. The charcteristic three-cysteine motif nucleates a [4Fe-4S] cluster, which binds SAM as a ligand to the unique Fe not ligated to a cysteine residue. The members participate in more than 40 distinct biochemical transformations, and most members have not been biochemically characterized. A handful of the members of this superfamily have been purified and at least partially characterized. Significant mechanistic and structural information is available for lysine 2,3-aminomutase, pyruvate formate-lyase, coproporphyrinogen III oxidase, and MoaA required for molybdopterin biosynthesis. Biochemical information is available for spore photoproduct lyase, anaerobic ribonucleotide reductase activation subunit, lipoyl synthase, and MiaB involved in methylthiolation of isopentenyladenine-37 in tRNA. The radical SAM enzymes biochemically characterized to date have in common the cleavage of the [4Fe-4S](1 +) -SAM complex to [4Fe-4S](2 +)-Met and the 5' -deoxyadenosyl radical, which abstracts a hydrogen atom from the substrate to initiate a radical mechanism.