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.     

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.     

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.     

Iron-sulfur cluster dynamics in biotin synthase: A new [2Fe-2S] cluster

Biotin synthase (BioB) catalyses the final step in the biosynthesis of biotin. Aerobically purified biotin synthase contains one [2Fe-2S]²⁺ cluster per monomer. However, active BioB contains in addition a [4Fe-4S]²⁺ cluster which can be formed either by reconstitution with iron and sulfide, or on reduction with sodium dithionite. Here, we use EPR spectroscopy to show that mutations in the conserved YNHNLD sequence of Escherichia coli BioB affect the formation and stability of the [4Fe-4S]¹⁺ cluster on reduction with dithionite and report the observation of a new [2Fe-2S]¹⁺ cluster. These results serve to illustrate the dynamic nature of iron-sulfur clusters in biotin synthase and the role played by the protein in cluster interconversion.     

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.     

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.     

Characterization of the cofactor composition of Escherichia coli biotin synthase

The cofactor content of in vivo, as-isolated, and reconstituted forms of recombinant Escherichia coli biotin synthase (BioB) has been investigated using the combination of UV-visible absorption, resonance Raman, and M?ssbauer spectroscopies along with parallel analytical and activity assays. In contrast to the recent report that E. coli BioB is a pyridoxal phosphate (PLP)-dependent enzyme with intrinsic cysteine desulfurase activity (Ollagnier-deChoudens, S., Mulliez, E., Hewitson, K. S., and Fontecave, M. (2002) Biochemistry 41, 9145-9152), no evidence for PLP binding or for PLP-induced cysteine desulfurase or biotin synthase activity was observed with any of the forms of BioB investigated in this work. The results confirm that BioB contains two distinct Fe-S cluster binding sites. One site accommodates a [2Fe-2S](2+) cluster with partial noncysteinyl ligation that can only be reconstituted in vitro in the presence of O(2). The other site accommodates a [4Fe-4S](2+,+) cluster that binds S-adenosylmethionine (SAM) at a unique Fe site of the [4Fe-4S](2+) cluster and undergoes O(2)-induced degradation via a distinct type of [2Fe-2S](2+) cluster intermediate. In vivo M?ssbauer studies show that recombinant BioB in anaerobically grown cells is expressed exclusively in an inactive form containing only the as-isolated [2Fe-2S](2+) cluster and demonstrate that the [2Fe-2S](2+) cluster is not a consequence of overexpressing the recombinant enzyme under aerobic growth conditions. Overall the results clarify the confusion in the literature concerning the Fe-S cluster composition and the in vitro reconstitution and O(2)-induced cluster transformations that are possible for recombinant BioB. In addition, they provide a firm foundation for assessing cluster transformations that occur during turnover and the catalytic competence of the [2Fe-2S](2+) cluster as the immediate S-donor for biotin biosynthesis.     

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.     

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.     

Loss of iron-sulfur clusters from biotin synthase as a result of catalysis promotes unfolding and degradation

Biotin synthase (BioB) is an S-adenosylmethionine radical enzyme that catalyzes addition of sulfur to dethiobiotin to form the biotin thiophane ring. In vitro, Escherichia coli BioB is active for only one turnover, during which the [2Fe-2S]²⁺ cluster is destroyed, one sulfide from the cluster is incorporated as the biotin thiophane sulfur, while Fe²⁺ ions and the remaining S²⁻ ion are released from the protein. The present work examines the fate of the protein following the loss of the FeS clusters. We examine the quaternary structure and thermal stability of active and inactive states of BioB, and find that loss of either the [4Fe-4S]²⁺ or [2Fe-2S]²⁺ clusters results in destabilization but not global unfolding of BioB. Using susceptibility to limited proteolysis as a guide, we find that specific regions of the protein appear to be transiently unfolded following loss of these clusters. We also examine the in vivo degradation of biotin synthase during growth in low-iron minimal media and find that BioB is degraded by an apparent ATP-dependent proteolysis mechanism that sequentially cleaves small fragments starting at the C-terminus. BioB appears to be resistant to degradation and capable of multiple turnovers only under high-iron conditions that favor repair of the FeS clusters, a process most likely mediated by the Isc or Suf iron-sulfur cluster assembly systems.     

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.     

Iron-sulfur center of biotin synthase and lipoate synthase

Biotin synthase and lipoate synthase are homodimers that are required for the C-S bond formation at nonactivated carbon in the biosynthesis of biotin and lipoic acid, respectively. Aerobically isolated monomers were previously shown to contain a (2Fe-2S) cluster, however, after incubation with dithionite one (4Fe-4S) cluster per dimer was obtained, suggesting that two (2Fe-2S) clusters had combined at the interface of the subunits to form the (4Fe-4S) cluster. Here we report M?ssbauer studies of (57)Fe-reconstituted biotin synthase showing that anaerobically prepared enzyme can accommodate two (4Fe-4S) clusters per dimer. The (4Fe-4S) cluster is quantitatively converted into a (2Fe-2S)(2+) cluster upon exposure to air. Reduction of the air-exposed enzyme with dithionite or photoreduced deazaflavin yields again (4Fe-4S) clusters. The (4Fe-4S) cluster is stable in both the 2+ and 1+ oxidation states. The M?ssbauer and EPR parameters were DeltaE(q) = 1.13 mm/s and delta = 0.44 mm/s for the diamagnetic (4Fe-4S)(2+) and DeltaE(q) = 0.51 mm/s, delta = 0.85 mm/s, g(par) = 2.035, and g(perp) = 1.93 for the S = (1)/(2) state of (4Fe-4S)(1+). Considering that we find two (4Fe-4S) clusters per dimer, our studies argue against the early proposal that the enzyme contains one (4Fe-4S) cluster bridging the two subunits. Our study of lipoate synthase gave results similar to those obtained for BS: under strict anaerobiosis, lipoate synthase can accommodate a (4Fe-4S) cluster per subunit [DeltaE(q) = 1.20 mm/s and delta = 0.44 mm/s for the diamagnetic (4Fe-4S)(2+) and g(par) = 2.039 and g(perp) = 1.93 for the S = (1)/(2) state of (4Fe-4S)(1+)], which reacts with oxygen to generate a (2Fe-2S)(2+) center.     

Mutagenesis of the proposed iron-sulfur cluster binding ligands in Escherichia coli biotin synthase

Biotin synthase (BioB) is a member of a family of enzymes that includes anaerobic ribonucleotide reductase and pyruvate formate lyase activating enzyme. These enzymes all use S-adenosylmethionine during turnover and contain three highly conserved cysteine residues that may act as ligands to an iron-sulfur cluster required for activity. Three mutant enzymes of BioB have been made, each with one cysteine residue (C53, 57, 60) mutated to alanine. All three mutant enzymes were inactive, but they still exhibited the characteristic UV-visible spectrum of a [2Fe-2S]2+ cluster similar to that of the wild-type enzyme.     

Fate of the (2Fe-2S)(2+) cluster of Escherichia coli biotin synthase during reaction: a Mössbauer characterization

Biotin synthase, the enzyme which catalyzes the last step of the biosynthesis of biotin, contains only (2Fe-2S)(2+) clusters when isolated under aerobic conditions. Previous results showed that reduction by dithionite or photoreduced deazaflavin converts the (2Fe-2S)(2+) to (4Fe-4S)(2+,+). However, until now, no detailed investigation concerning the fate of the (2Fe-2S)(2+) during reduction under assay conditions (NADPH, flavodoxin, flavodoxin reductase) has been realized. Here, we show by M?ssbauer spectroscopy on a partially purified fraction overexpressing the enzyme that, in the presence of a S(2)(-) source and Fe(2+), there is conversion of the predominant (2Fe-2S)(2+) clusters into a 1:1 mixture of (2Fe-2S)(2+) and (4Fe-4S)(2+). No change in this cluster composition was observed in the presence of the physiological reducing system. When the reaction was allowed to proceed by addition of the substrate dethiobiotin, the (4Fe-4S)(2+) was untouched whereas the (2Fe-2S)(2+) was degraded into a new species. This is consistent with the hypothesis that the reduced (4Fe-4S) cluster is involved in mediating the cleavage of AdoMet and that the (2Fe-2S)(2+) is the sulfur source for biotin.     

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.     

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.     

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.     

The iron-sulfur center of biotin synthase: site-directed mutants

Biotin synthase contains an essential [4Fe-4S]+ cluster that is thought to provide an electron for the cleavage of S-adenosylmethionine, a cofactor required for biotin formation. The conserved cysteine residues Cys53, Cys57 and Cys60 have been proposed as ligands to the [4Fe-4S] cluster. These residues belong to a C-X3-C-X2-C motif which is also found in pyruvate formate lyase-activating enzyme, lysine 2,3-aminomutase and the anaerobic ribonucleotide reductase-activating component. To investigate the role of the cysteine residues, Cys-->Ala mutants of the eight cysteine residues of Escherichia coli biotin synthase were prepared and assayed for activity. Our results show that six cysteines are important for biotin formation. Only two mutant proteins, C276A and C288A, closely resembled the wild-type protein, indicating that the corresponding cysteines are not involved in iron chelation and biotin formation. The six other mutant proteins, C53A, C57A, C60A, C97A, C128A and C188A, were inactive but capable of assembling a [4Fe-4S] cluster, as shown by M?ssbauer spectroscopy. The C53A, C57A and C60A mutant proteins are unique in that their cluster could not undergo reduction to the [4Fe-4S]+ state, as shown by EPR and M?ssbauer spectroscopy. On this basis and by analogy with pyruvate formate lyase-activating enzyme and the anaerobic ribonucleotide reductase-activating component, it is suggested that the corresponding cysteines coordinate the cluster even though one cannot fully exclude the possibility that other cysteines play that role as well. Therefore it appears that for activity biotin synthase absolutely requires cysteines that are not involved in iron chelation.     

Recombinant Escherichia coli biotin synthase is a [2Fe-2S](2+) protein in whole cells

EPR and M?ssbauer spectroscopies have been used to determine the type and properties of the iron-sulfur clusters present in homologously expressed recombinant Escherichia coli BioB in whole cells prior to purification. Difference EPR spectra of samples of whole cells from a strain over-expressing E. coli BioB and a strain containing the same plasmid but without the bioB insertion showed an axial S=1/2 resonance that was attributed to the [2Fe-2S](+) cluster of the E. coli iron-sulfur cluster assembly 2Fe ferredoxin, based on principal g-values, linewidths and relaxation behavior. Comparison of the M?ssbauer spectra of whole cells with and without the bioB insertion revealed that the E. coli cells with over-expressed BioB contain an additional species that exhibits a spectrum identical to that of the [2Fe-2S](2+) cluster in purified recombinant BioB. The concentration of this [2Fe-2S](2+) species in the whole cell sample was quantified using a M?ssbauer standard and found to be approximately 260 microM, which was comparable to the BioB protein concentration estimated for the cell paste. The results demonstrate that the [2Fe-2S](2+) cluster found in purified samples of recombinant BioB is not an artifact of the protein purification procedure, and indicate that recombinant BioB is over-expressed in an inactive form during aerobic growth.