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.     

Characterization of an active spore photoproduct lyase, a DNA repair enzyme in the radical S-adenosylmethionine superfamily

The major photoproduct in UV-irradiated Bacillus spore DNA is a unique thymine dimer called spore photoproduct (SP, 5-thyminyl-5,6-dihydrothymine). The enzyme spore photoproduct lyase (SP lyase) has been found to catalyze the repair of SP dimers to thymine monomers in a reaction that requires S-adenosylmethionine. We present here the first detailed characterization of catalytically active SP lyase, which has been anaerobically purified from overexpressing Escherichia coli. Anaerobically purified SP lyase is monomeric and is red-brown in color. The purified enzyme contains approximately 3.1 iron and 3.0 acid-labile S(2-) per protein and has a UV-visible spectrum characteristic of iron-sulfur proteins (410 nm (11.9 mM(-1) cm(-1)) and 450 nm (10.5 mM(-1) cm(-1))). The X-band EPR spectrum of the purified enzyme shows a nearly isotropic signal (g = 2.02) characteristic of a [3Fe-4S]1+ cluster; reduction of SP lyase with dithionite results in the appearance of a new EPR signal (g = 2.03, 1.93, and 1.89) with temperature dependence and g values consistent with its assignment to a [4Fe-4S]1+ cluster. The reduced purified enzyme is active in SP repair, with a specific activity of 0.33 micromol/min/mg. Only a catalytic amount of S-adenosylmethionine is required for DNA repair, and no irreversible cleavage of S-adenosylmethionine into methionine and 5'-deoxyadenosine is observed during the reaction. Label transfer from [5'-3H]S-adenosylmethionine to repaired thymine is observed, providing evidence to support a mechanism in which a 5'-deoxyadenosyl radical intermediate directly abstracts a hydrogen from SP C-6 to generate a substrate radical, and subsequent to radical-mediated beta-scission, a product thymine radical abstracts a hydrogen from 5'-deoxyadenosine to regenerate the 5'-deoxyadenosyl radical. Together, our results support a mechanism in which S-adenosylmethionine acts as a catalytic cofactor, not a substrate, in the DNA repair reaction.     

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.     

Characterization of NocL involved in thiopeptide nocathiacin I biosynthesis: a [4Fe-4S] cluster and the catalysis of a radical S-adenosylmethionine enzyme

The radical S-adenosylmethionine (AdoMet) enzyme superfamily is remarkable at catalyzing chemically diverse and complex reactions. We have previously shown that NosL, which is involved in forming the indole side ring of the thiopeptide nosiheptide, is a radical AdoMet enzyme that processes L-Trp to afford 3-methyl-2-indolic acid (MIA) via an unusual fragmentation-recombination mechanism. We now report the expansion of the MIA synthase family by characterization of NocL, which is involved in nocathiacin I biosynthesis. EPR and UV-visible absorbance spectroscopic analyses demonstrated the interaction between L-Trp and the [4Fe-4S] cluster of NocL, leading to the assumption of nonspecific interaction of [4Fe-4S] cluster with other nucleophiles via the unique Fe site. This notion is supported by the finding of the heterogeneity in the [4Fe-4S] cluster of NocL in the absence of AdoMet, which was revealed by the EPR study at very low temperature. Furthermore, a free radical was observed by EPR during the catalysis, which is in good agreement with the hypothesis of a glycyl radical intermediate. Combined with the mutational analysis, these studies provide new insights into the function of the [4Fe-4S] cluster of radical AdoMet enzymes as well as the mechanism of the radical-mediated complex carbon chain rearrangement catalyzed by MIA synthase.     

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.     

Control of adenosylmethionine-dependent radical generation in biotin synthase: a kinetic and thermodynamic analysis of substrate binding to active and inactive forms of BioB

Biotin synthase (BS) is an AdoMet-dependent radical enzyme that catalyzes the insertion of sulfur into saturated C6 and C9 atoms in the substrate dethiobiotin. To facilitate sulfur insertion, BS catalyzes the reductive cleavage of AdoMet to methionine and 5'-deoxyadenosyl radicals, which then abstract hydrogen atoms from the C6 and C9 positions of dethiobiotin. The enzyme from Escherichia coli is purified as a dimer that contains one [2Fe-2S]2+ cluster per monomer and can be reconstituted in vitro to contain an additional [4Fe-4S]2+ cluster per monomer. Since each monomer contains each type of cluster, the dimeric enzyme could contain one active site per monomer, or could contain a single active site at the dimer interface. To address these possibilities, and to better understand the manner in which biotin synthase controls radical generation and reactivity, we have examined the binding of AdoMet and DTB to reconstituted biotin synthase. We find that both the [2Fe-2S]2+ cluster and the [4Fe-4S]2+ cluster must be present for tight substrate binding. Further, substrate binding is highly cooperative, with the affinity for AdoMet increasing >20-fold in the presence of DTB, while DTB binds only in the presence of AdoMet. The stoichiometry of binding is ca. 2:1:1 AdoMet:DTB:BS dimer, suggesting that biotin synthase has a single functional active site per dimer. AdoMet binding, either in the presence or in the absence of DTB, leads to a decrease in the magnitude of the UV-visible absorption band at approximately 400 nm that we attribute to changes in the coordination environment of the [4Fe-4S]2+ cluster. Using these spectral changes as a probe, we have examined the kinetics of AdoMet and DTB binding, and propose an ordered binding mechanism that is followed by a conformational change in the enzyme-substrate complex. This kinetic analysis suggests that biotin synthase is evolved to bind AdoMet both weakly and slowly in the absence of DTB, while both the rate of binding and the affinity for AdoMet are increased in the presence of DTB. Cooperative binding of AdoMet and DTB may be an important mechanism for limiting the production of 5'-deoxyadenosyl radicals in the absence of the correct substrate.     

Electron paramagnetic resonance evidence for a novel interconversion of [3Fe-4S](+) and [4Fe-4S](+) clusters with endogenous iron and sulfide in anaerobic ribonucleotide reductase activase in vitro

We report an EPR study of the iron-sulfur enzyme, anaerobic ribonucleotide reductase activase from Lactococcus lactis. The activase (nrdG gene) together with S-adenosyl-L-methionine (AdoMet) give rise to a glycyl radical in the NrdD component. A semi-reduced [4Fe-4S](+) cluster with an axially symmetric EPR signal was produced upon photochemical reduction of the activase. Air exposure of the reduced enzyme gave a [3Fe-4S](+) cluster. The Fe(3)S(4) cluster was convertible to the EPR-active [4Fe-4S](+) cluster by renewed treatment with reducing agents, demonstrating a reversible [3Fe-4S](+)- to-[4Fe-4S](+) cluster conversion without exogenous addition of iron or sulfide. Anaerobic reduction of the activase by a moderate concentration of dithionite also resulted in a semi-reduced [4Fe-4S](+) cluster. Prolonged reduction gave an EPR-silent fully reduced state, which was enzymatically inactive. Both reduced states gave the [3Fe-4S](+) EPR signal after air exposure. The iron-sulfur cluster interconversion was also studied in the presence of AdoMet. The EPR signal of semi-reduced activase-AdoMet had rhombic symmetry and was independent of which reductant was applied, whereas the EPR signal of the [3Fe-4S](+) cluster after air exposure was unchanged. The results indicate that an AdoMet-mediated [4Fe-4S](+) center is the native active species that induces the formation of a glycyl radical in the NrdD component.     

Characterization of MOCS1A, an oxygen-sensitive iron-sulfur protein involved in human molybdenum cofactor biosynthesis

The human proteins MOCS1A and MOCS1B catalyze the conversion of a guanosine derivative to precursor Z during molybdenum cofactor biosynthesis. MOCS1A shares homology with S-adenosylmethionine (AdoMet)-dependent radical enzymes, which catalyze the formation of protein and/or substrate radicals by reductive cleavage of AdoMet through a [4Fe-4S] cluster. Sequence analysis of MOCS1A showed two highly conserved cysteine motifs, one near the N terminus and one near the C terminus. MOCS1A was heterologously expressed in Escherichia coli and purified under aerobic and anaerobic conditions. Individual mutations of the conserved cysteines to serine revealed that all are essential for synthesis of precursor Z in vivo. The type and properties of the iron-sulfur (FeS) clusters were investigated using a combination of UV-visible absorption, variable temperature magnetic circular dichroism, resonance Raman, M?ssbauer, and EPR spectroscopies coupled with iron and acid-labile sulfide analyses. The results indicated that anaerobically purified MOCS1A is a monomeric protein containing two oxygen-sensitive FeS clusters, each coordinated by only three cysteine residues. A redox-active [4Fe-4S](2+,+) cluster is ligated by an N-terminal CX(3)CX(2)C motif as is the case with all other AdoMet-dependent radical enzymes investigated thus far. A C-terminal CX(2)CX(13)C motif that is unique to MOCS1A and its orthologs primarily ligates a [3Fe-4S](0) cluster. However, MOCS1A could be reconstituted in vitro under anaerobic conditions to yield a form containing two [4Fe-4S](2+) clusters. The N-terminal [4Fe-4S](2+) cluster was rapidly degraded by oxygen via a semistable [2Fe-2S](2+) cluster intermediate, and the C-terminal [4Fe-4S](2+) cluster was rapidly degraded by oxygen to yield a semistable [3Fe-4S](0) cluster intermediate.     

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.     

Enzymatic modification of tRNAs: MiaB is an iron-sulfur protein

The product of the miaB gene, MiaB, from Escherichia coli participates in the methylthiolation of the adenosine 37 residue during modification of tRNAs that read codons beginning with uridine. A His-tagged version of MiaB has been overproduced and purified to homogeneity. Gel electrophoresis and size exclusion chromatography revealed that MiaB protein is a monomer. As isolated MiaB contains both iron and sulfide and an apoprotein form can chelate as much as 2.5-3 iron and 3-3.5 sulfur atoms per polypeptide chain. UV-visible and EPR spectroscopy of MiaB indicate the presence of a [4Fe-4S] cluster under reducing and anaerobic conditions, whereas [2Fe-2S] and [3Fe-4S] forms are generated under aerobic conditions. Preliminary site-directed mutagenesis studies suggest that Cys(157), Cys(161), and Cys(164) are involved in iron chelation and that the cluster is essential for activity. Together with the previously shown requirement of S-adenosylmethionine (AdoMet) for the methylthiolation reaction, the finding that MiaB is an iron-sulfur protein suggests that it belongs to a superfamily of enzymes that uses [Fe-S] centers and AdoMet to initiate radical catalysis. MiaB is the first and only tRNA modification enzyme known to contain an Fe-S cluster.     

Cysteine Methylation Controls Radical Generation in the Cfr Radical AdoMet rRNA Methyltransferase

The ‘radical S-adenosyl-L-methionine (AdoMet)’ enzyme Cfr methylates adenosine 2503 of the 23S rRNA in the peptidyltransferase centre (P-site) of the bacterial ribosome. This modification protects host bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA), from numerous antibiotics, including agents (e.g. linezolid, retapamulin) that were developed to treat such organisms. Cfr contains a single [4Fe-4S] cluster that binds two separate molecules of AdoMet during the reaction cycle. These are used sequentially to first methylate a cysteine residue, Cys338; and subsequently generate an oxidative radical intermediate that facilitates methyl transfer to the unreactive C8 (and/or C2) carbon centres of adenosine 2503. How the Cfr active site, with its single [4Fe-4S] cluster, catalyses these two distinct activities that each utilise AdoMet as a substrate remains to be established. Here, we use absorbance and electron paramagnetic resonance (EPR) spectroscopy to investigate the interactions of AdoMet with the [4Fe-4S] clusters of wild-type Cfr and a Cys338 Ala mutant, which is unable to accept a methyl group. Cfr binds AdoMet with high (∼ 10 µM) affinity notwithstanding the absence of the RNA cosubstrate. In wild-type Cfr, where Cys338 is methylated, AdoMet binding leads to rapid oxidation of the [4Fe-4S] cluster and production of 5''-deoxyadenosine (DOA). In contrast, while Cys338 Ala Cfr binds AdoMet with equivalent affinity, oxidation of the [4Fe-4S] cluster is not observed. Our results indicate that the presence of a methyl group on Cfr Cys338 is a key determinant of the activity of the enzyme towards AdoMet, thus enabling a single active site to support two distinct modes of AdoMet cleavage.     

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.     

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.     

Role of the [2Fe-2S]2+ cluster in biotin synthase: mutagenesis of the atypical metal ligand arginine 260

Biotin synthase (BS) is an S-adenosylmethionine (AdoMet)-dependent radical enzyme that catalyzes the addition of sulfur to dethiobiotin. Like other AdoMet radical enzymes, BS contains a [4Fe-4S] cluster that is coordinated by a highly conserved CxxxCxxC sequence motif and by the methionyl amine and carboxylate of AdoMet. The close association of the [4Fe-4S]+ cluster with AdoMet facilitates reductive cleavage of the sulfonium and the generation of transient 5'-deoxyadenosyl radicals, which are then proposed to sequentially abstract hydrogen atoms from the substrate to produce carbon radicals at C9 and C6 of dethiobiotin. BS also contains a [2Fe-2S]2+ cluster located approximately 4-5 A from dethiobiotin, and we have proposed that a bridging sulfide of this cluster quenches the substrate radicals, leading to formation of the thiophane ring of biotin. In BS from Escherichia coli, the [2Fe-2S]2+ cluster is coordinated by cysteines 97, 128, and 188, and the atypical metal ligand, arginine 260. The evolutionary conservation of an arginine guanidinium as a metal ligand suggests a novel role for this residue in tuning the reactivity or stability of the [2Fe-2S]2+ cluster. In this work, we explore the effects of mutagenesis of Arg260 to Ala, Cys, His, and Met. Although perturbations in a number of characteristics of the [2Fe-2S]2+ cluster and the proteins are noted, the reconstituted enzymes have in vitro single-turnover activities that are 30-120% of that of the wild type. Further, in vivo expression of each mutant enzyme was sufficient to sustain growth of a bioB- mutant strain on dethiobiotin-supplemented medium, suggesting the enzymes were active and efficiently reconstituted by the in vivo iron-sulfur cluster (ISC) assembly system. Although we cannot exclude an as-yet-unidentified in vivo role in cluster repair or retention, we can conclude that Arg260 is not essential for the catalytic reaction of BS.     

DNA repair and free radicals, new insights into the mechanism of spore photoproduct lyase revealed by single amino acid substitution

The major DNA photoproduct in UV-irradiated Bacillus subtilis spores is the thymine dimer named spore photoproduct (SP, 5-(alpha-thyminyl)-5,6-dihydrothymine). The SP lesion has been found to be efficiently repaired by SP lyase (SPL) a very specific enzyme that reverses the SP to two intact thymines, at the origin of the great resistance of the spores to UV irradiation. SPL belongs to a superfamily of [4Fe-4S] iron-sulfur enzymes, called "Radical-SAM." Here, we show that the single substitution of cysteine 141 into alanine, a residue fully conserved in Bacillus species and previously shown to be essential for spore DNA repair in vivo, has a major impact on the outcome of the SPL-dependent repair reaction in vitro. Indeed the modified enzyme catalyzes the almost quantitative conversion of the SP lesion into one thymine and one thymine sulfinic acid derivative. This compound results from the trapping of the allyl-type radical intermediate by dithionite, used as reducing agent in the reaction mixture. Implications of the data reported here regarding the repair mechanism and the role of Cys-141 are discussed.     

Essential Cysteine Residues in Bacillus subtilis Spore Photoproduct Lyase Identified by Alanine Scanning Mutagenesis

Endospore-forming bacteria (Bacillus and Clostridium spp.) are highly ultraviolet (UV) resistant and repair UV-induced DNA damage in part using the spore-specific DNA repair enzyme spore photoproduct (SP) lyase. SP lyase in all known sporeformers contains four conserved cysteine residues; three absolutely conserved residues are located at the “Radical SAM” consensus (C91xxxC95xxC98), which presumably participates in [4Fe-4S] cluster formation. A fourth conserved cysteine, the function of which is unknown, is located at C141 in SP lyase from all Bacillus spp. sequenced to date. To probe the function of the fourth cysteine, each conserved cysteine in the B. subtilis SP lyase was systematically altered to alanine by site-directed mutagenesis. UV-visible spectroscopy of wild-type and mutant SP lyases indicated that C141 does not participate in [4Fe-4S] formation and redox chemistry; however, in vivo SP lyase activity was abolished in all mutants, indicating an essential role for C141 in SP lyase activity.     

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.     

IscA, an alternate scaffold for Fe-S cluster biosynthesis.

An IscA homologue within the nif regulon of Azotobacter vinelandii, designated (Nif)IscA, was expressed in Escherichia coli and purified to homogeneity. Purified (Nif)IscA was found to be a homodimer of 11-kDa subunits that contained no metal centers or other prosthetic groups in its as-isolated form. Possible roles for (Nif)IscA in Fe-S cluster biosynthesis were assessed by investigating the ability to bind iron and to assemble Fe-S clusters in a NifS-directed process, as monitored by the combination of UV-vis absorption, M?ssbauer, resonance Raman, variable-temperature magnetic circular dichroism, and EPR spectroscopies. Although (Nif)IscA was found to bind ferrous ion in a tetrahedral, predominantly cysteinyl-ligated coordination environment, the low-binding affinity argues against a specific role as a metallochaperone for the delivery of ferrous ion to other Fe-S cluster assembly proteins. Rather, a role for (Nif)IscA as an alternate scaffold protein for Fe-S cluster biosynthesis is proposed, based on the NifS-directed assembly of approximately one labile [4Fe-4S](2+) cluster per (Nif)IscA homodimer, via a transient [2Fe-2S](2+) cluster intermediate. The cluster assembly process was monitored temporally using UV-vis absorption and M?ssbauer spectroscopy, and the intermediate [2Fe-2S](2+)-containing species was additionally characterized by resonance Raman spectroscopy. The M?ssbauer and resonance Raman properties of the [2Fe-2S](2+) center are consistent with complete cysteinyl ligation. The presence of three conserved cysteine residues in all IscA proteins and the observed cluster stoichiometry of approximately one [2Fe-2S](2+) or one [4Fe-4S](2+) per homodimer suggest that both cluster types are subunit bridging. In addition, (Nif)IscA was shown to couple delivery of iron and sulfur by using ferrous ion to reduce sulfane sulfur. The ability of Fe-S scaffold proteins to couple the delivery of these two toxic and reactive Fe-S cluster precursors is likely to be important for minimizing the cellular concentrations of free ferrous and sulfide ions. On the basis of the spectroscopic and analytical results, mechanistic schemes for NifS-directed cluster assembly on (Nif)IscA are proposed. It is proposed that the IscA family of proteins provide alternative scaffolds to the NifU and IscU proteins for mediating nif-specific and general Fe-S cluster assembly.     

Selective oxidative destruction of iron-sulfur clusters. Ferricyanide oxidation of Azotobacter vinelandii ferredoxin I

The destructive oxidation of aerobically isolated 7Fe Azotobacter vinelandii ferredoxin I [(7Fe)FdI] by Fe(CN)3-6 is examined using low-temperature magnetic circular dichroism (MCD) and EPR. The results demonstrate that oxidation of the [3Fe-3S] cluster occurs only after essentially complete destruction of the [4Fe-4S] cluster. It is therefore feasible by controlled Fe(CN)3-6 oxidation to obtain a partially metallated form of FdI, (3Fe)FdI, containing only a [3Fe-3S] cluster. The MCD and EPR data demonstrate that the [3Fe-3S] cluster in (3Fe)FdI is essentially identical in structure to that in the native protein.     

[4Fe-4S] cluster trafficking mediated by Arabidopsis mitochondrial ISCA and NFU proteins

Numerous iron-sulfur (Fe-S) proteins with diverse functions are present in the matrix and respiratory chain complexes of mitochondria. Although [4Fe-4S] clusters are the most common type of Fe-S cluster in mitochondria, the molecular mechanism of [4Fe-4S] cluster assembly and insertion into target proteins by the mitochondrial iron-sulfur cluster (ISC) maturation system is not well-understood. Here we report a detailed characterization of two late-acting Fe-S cluster-carrier proteins from Arabidopsis thaliana, NFU4 and NFU5. Yeast two-hybrid and bimolecular fluorescence complementation studies demonstrated interaction of both the NFU4 and NFU5 proteins with the ISCA class of Fe-S carrier proteins. Recombinant NFU4 and NFU5 were purified as apo-proteins after expression in Escherichia coli. In vitro Fe-S cluster reconstitution led to the insertion of one [4Fe-4S]²⁺ cluster per homodimer as determined by UV-visible absorption/CD, resonance Raman and EPR spectroscopy, and analytical studies. Cluster transfer reactions, monitored by UV-visible absorption and CD spectroscopy, showed that a [4Fe-4S]²⁺ cluster-bound ISCA1a/2 heterodimer is effective in transferring [4Fe-4S]²⁺ clusters to both NFU4 and NFU5 with negligible back reaction. In addition, [4Fe-4S]²⁺ cluster-bound ISCA1a/2, NFU4, and NFU5 were all found to be effective [4Fe-4S]²⁺ cluster donors for maturation of the mitochondrial apo-aconitase 2 as assessed by enzyme activity measurements. The results demonstrate rapid, unidirectional, and quantitative [4Fe-4S]²⁺ cluster transfer from ISCA1a/2 to NFU4 or NFU5 that further delineates their respective positions in the plant ISC machinery and their contributions to the maturation of client [4Fe-4S] cluster-containing proteins.