The Radical SAM Superfamily

ABSTRACT

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.     

S-adenosylmethionine as an oxidant: the radical SAM superfamily

A recently discovered superfamily of enzymes function using chemically novel mechanisms, in which S-adenosylmethionine (SAM) serves as an oxidizing agent in DNA repair and the biosynthesis of vitamins, coenzymes and antibiotics. Members of this superfamily, the radical SAM enzymes, are related by the cysteine motif CxxxCxxC, which nucleates the [4Fe-4S] cluster found in each. A common thread in the novel chemistry of these proteins is the use of a strong reducing agent--a low-potential [4Fe-4S](1+) cluster--to generate a powerful oxidizing agent, the 5'-deoxyadenosyl radical, from SAM. Recent results are beginning to determine the unique biochemistry for some of the radical SAM enzymes, for example, lysine 2,3 aminomutase, pyruvate formate lyase activase and biotin synthase.     

Cofactor dependence of reduction potentials for [4Fe-4S]2+/1+ in lysine 2,3-aminomutase

Lysine 2,3-aminomutase (LAM) catalyzes the interconversion of l-lysine and l-beta-lysine by a free radical mechanism. The 5'-deoxyadenosyl radical derived from the reductive cleavage of S-adenosyl-l-methionine (SAM) initiates substrate-radical formation. The [4Fe-4S](1+) cluster in LAM is the one-electron source in the reductive cleavage of SAM, which is directly ligated to the unique iron site in the cluster. We here report the midpoint reduction potentials of the [4Fe-4S](2+/1+) couple in the presence of SAM, S-adenosyl-l-homocysteine (SAH), or 5'-{N-[(3S)-3-aminocarboxypropyl]-N-methylamino}-5'-deoxyadenosine (azaSAM) as measured by spectroelectrochemistry. The reduction potentials are -430 +/- 2 mV in the presence of SAM, -460 +/- 3 mV in the presence of SAH, and -497 +/- 10 mV in the presence of azaSAM. In the absence of SAM or an analogue and the presence of dithiothreitol, dihydrolipoate, or cysteine as ligands to the unique iron, the midpoint potentials are -479 +/- 5, -516 +/- 5, and -484 +/- 3 mV, respectively. LAM is a member of the radical SAM superfamily of enzymes, in which the CxxxCxxC motif donates three thiolate ligands to iron in the [4Fe-4S] cluster and SAM donates the alpha-amino and alpha-carboxylate groups of the methionyl moiety as ligands to the fourth iron. The results show the reduction potentials in the midrange for ferredoxin-like [4Fe-4S] clusters. They show that SAM elevates the reduction potential by 86 mV relative to that of dihydrolipoate as the cluster ligand. This difference accounts for the SAM-dependent reduction of the [4Fe-4S](2+) cluster by dithionite reported earlier. Analogues of SAM have a weakened capacity to raise the potential. We conclude that the midpoint reduction potential of the cluster ligated to SAM is 1.2 V less negative than the half-wave potential for the one-electron reductive cleavage of simple alkylsulfonium ions in aqueous solution. The energetic barrier in the reductive cleavage of SAM may be overcome through the use of binding energy.     

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.     

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.     

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.     

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.     

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 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.     

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.     

Adenosyl coenzyme and pH dependence of the [4Fe-4S]2+/1+ transition in lysine 2,3-aminomutase

5'-[N-[(3S)-3-Amino-carboxypropyl]-N-methylamino]-5(')-deoxyadenosine (azaSAM), an analog of S-adenosyl-L-methionine (SAM), was used to study the cofactor-dependent reduction of the [4Fe-4S](2+) center in lysine 2,3-aminomutase to the +1 oxidation state. azaSAM has a tertiary nitrogen in place of the sulfonium center of SAM. The analog binds to lysine 2,3-aminomutase with K(d)s of 1.4+/-0.3 microM at pH 8.0 and 2.2+/-0.6 microM at pH 6.5. Reduction of the [4Fe-4S](2+) center in the presence of this analog gives a 10K [4Fe-4S](1+) electron paramagnetic resonance (EPR) signal similar to that seen with SAM or S-adenosyl-L-homocysteine (SAH). The pH dependence of cofactor-induced reduction was examined to determine whether ionization of the tertiary nitrogen (pK(a)=7.08) might affect reduction of the [4Fe-4S](2+) center. The results show similar behavior in azaSAM and SAH, demonstrating that ionization of the aza group in azaSAM does not account for pH dependence in cofactor-dependent reduction of the [4Fe-4S](2+) center. The signal shape of the low-temperature EPR signal for the [4Fe-4S](1+) center in the SAM-induced reduction displayed a pH dependence that was not observed in the azaSAM- or SAH-induced spectra. Unique features of the signal are at a maximum at the pH activity optimum of pH 8 and are diminished as the pH is lowered or raised. These features are also absent in the spectra at all pHs examined when reduction is induced by azaSAM or SAH.     

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.     

Pyruvate formate-lyase activating enzyme: elucidation of a novel mechanism for glycyl radical formation

Pyruvate formate lyase activating enzyme is a member of a novel superfamily of enzymes that utilize S-adenosylmethionine to initiate radical catalysis. This enzyme has been isolated with several different iron-sulfur clusters, but single turnover monitored by EPR has identified the [4Fe-4S](1+) cluster as the catalytically active cluster; this cluster is believed to be oxidized to the [4Fe-4S](2+) state during turnover. The [4Fe-4S] cluster is coordinated by a three-cysteine motif common to the radical/S-adenosylmethionine superfamily, suggesting the presence of a unique iron in the cluster. The unique iron site has been confirmed by Mossbauer and ENDOR spectroscopy experiments, which also provided the first evidence for direct coordination of S-adenosylmethionine to an iron-sulfur cluster, in this case the unique iron of the [4Fe-4S] cluster. Coordination to the unique iron anchors the S-adenosylmethionine in the active site, and allows for a close association between the sulfonium of S-adenosylmethionine and the cluster as observed by ENDOR spectroscopy. The evidence to date leads to a mechanistic proposal involving inner-sphere electron transfer from the cluster to the sulfonium of S-adenosylmethionine, followed by or concomitant with C-S bond homolysis to produce a 5'-deoxyadenosyl radical; this transient radical abstracts a hydrogen atom from G734 to activate pyruvate formate lyase.     

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.     

Escherichia coli lipoyl synthase binds two distinct [4Fe-4S] clusters per polypeptide

Lipoyl synthase (LS) is a member of a recently established class of metalloenzymes that use S-adenosyl-l-methionine (SAM) as the precursor to a high-energy 5'-deoxyadenosyl 5'-radical (5'-dA(*)). In the LS reaction, the 5'-dA(*) is hypothesized to abstract hydrogen atoms from C-6 and C-8 of protein-bound octanoic acid with subsequent sulfur insertion, generating the lipoyl cofactor. Consistent with this premise, 2 equiv of SAM is required to synthesize 1 equiv of the lipoyl cofactor, and deuterium transfer from octanoyl-d(15) H-protein of the glycine cleavage system-one of the substrates for LS-has been reported [Cicchillo, R. M., Iwig, D. F., Jones, A. D., Nesbitt, N. M., Baleanu-Gogonea, C., Souder, M. G., Tu, L., and Booker, S. J. (2004) Biochemistry 43, 6378-6386]. However, the exact identity of the sulfur donor remains unknown. We report herein that LS from Escherichia coli can accommodate two [4Fe-4S] clusters per polypeptide and that this form of the enzyme is relevant to turnover. One cluster is ligated by the cysteine amino acids in the C-X(3)-C-X(2)-C motif that is common to all radical SAM enzymes, while the other is ligated by the cysteine amino acids residing in a C-X(4)-C-X(5)-C motif, which is conserved only in lipoyl synthases. When expressed in the presence of a plasmid that harbors an Azotobacter vinelandii isc operon, which is involved in Fe/S cluster biosynthesis, the as-isolated wild-type enzyme contained 6.9 +/- 0.5 irons and 6.4 +/- 0.9 sulfides per polypeptide and catalyzed formation of 0.60 equiv of 5'-deoxyadenosine (5'-dA) and 0.27 equiv of lipoylated H-protein per polypeptide. The C68A-C73A-C79A triple variant, expressed and isolated under identical conditions, contained 3.0 +/- 0.1 irons and 3.6 +/- 0.4 sulfides per polypeptide, while the C94A-C98A-C101A triple variant contained 4.2 +/- 0.1 irons and 4.7 +/- 0.8 sulfides per polypeptide. Neither of these variant proteins catalyzed formation of 5'-dA or the lipoyl group. M?ssbauer spectroscopy of the as-isolated wild-type protein and the two triple variants indicates that greater than 90% of all associated iron is in the configuration [4Fe-4S](2+). When wild-type LS was reconstituted with (57)Fe and sodium sulfide, it harbored considerably more iron (13.8 +/- 0.6) and sulfide (13.1 +/- 0.2) per polypeptide and catalyzed formation of 0.96 equiv of 5'-dA and 0.36 equiv of the lipoyl group. M?ssbauer spectroscopy of this protein revealed that only approximately 67% +/- 6% of the iron is in the form of [4Fe-4S](2+) clusters, amounting to 9.2 +/- 0.4 irons and 8.8 +/- 0.1 sulfides or 2 [4Fe-4S](2+) clusters per polypeptide, with the remainder of the iron occurring as adventitiously bound species. Although the M?ssbauer parameters of the clusters associated with each of the variants are similar, EPR spectra of the reduced forms of the cluster show small differences in spin concentration and g-values, consistent with each of these clusters as distinct species residing in each of the two cysteine-containing motifs.     

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.     

Mechanistic understanding of Pyrococcus horikoshii Dph2, a [4Fe-4S] enzyme required for diphthamide biosynthesis

Diphthamide, the target of diphtheria toxin, is a unique posttranslational modification on eukaryotic and archaeal translation elongation factor 2 (EF2). The proposed biosynthesis of diphthamide involves three steps and we have recently found that in Pyrococcus horikoshii (P. horikoshii), the first step uses an S-adenosyl-L-methionine (SAM)-dependent [4Fe-4S] enzyme, PhDph2, to catalyze the formation of a C-C bond. Crystal structure shows that PhDph2 is a homodimer and each monomer contains three conserved cysteine residues that can bind a [4Fe-4S] cluster. In the reduced state, the [4Fe-4S] cluster can provide one electron to reductively cleave the bound SAM molecule. However, different from classical radical SAM family of enzymes, biochemical evidence suggest that a 3-amino-3-carboxypropyl radical is generated in PhDph2. Here we present evidence supporting that the 3-amino-3-carboxypropyl radical does not undergo hydrogen abstraction reaction, which is observed for the deoxyadenosyl radical in classical radical SAM enzymes. Instead, the 3-amino-3-carboxypropyl radical is added to the imidazole ring in the pathway towards the formation of the product. Furthermore, our data suggest that the chemistry requires only one [4Fe-4S] cluster to be present in the PhDph2 dimer.