Biochimica et Biophysica Acta 1824 (2012) 1213–1222

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Review

Biotin synthase: Insights into radical-mediated carbon–sulfur bond formation☆

Corey J. Fugate, Joseph T. Jarrett ⁎Department of Chemistry, University of Hawaii at Manoa, Honolulu, HI 96822, USA

Abbreviations: ACP, acyl carrier protein; AdoHcyAdoMet, S-adenosyl-L-methionine; AON, 8-amino-7-oA; DAPA, 7,8-diaminopelargonic acid; DAPA, 7,8-diaminoptin or 5-methyl-2-oxo-4-imidazolidinehexanoic acid; ENresonance; EPR, electron paramagnetic resonance; ESEEMmodulation; 9-MDTB, 9-mercaptodethiobiotin; MTAN, 5′5′-dAH nucleosidase; PLP, pyridoxal phosphate☆ This article is part of a Special Issue entitled: RadiEnzymology.⁎ Corresponding author at: 2545 McCarthy Mall, Bil

2233, USA. Tel.: +1 808 956 6721; fax: +1 808 956 59E-mail address: jtj@hawaii.edu (J.T. Jarrett).

1570-9639/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.bbapap.2012.01.010

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 October 2011Accepted 17 January 2012Available online 28 January 2012

Keywords:Enzyme mechanismRadical enzymeIron-sulfur clusterBiotin biosynthesisCofactor biosynthesis

The enzyme cofactor and essential vitamin biotin is biosynthesized in bacteria, fungi, and plants through apathway that culminates with the addition of a sulfur atom to generate the five-membered thiophane ring.The immediate precursor, dethiobiotin, has methylene and methyl groups at the C6 and C9 positions, respec-tively, and formation of a thioether bridging these carbon atoms requires cleavage of unactivated C\H bonds.Biotin synthase is an S-adenosyl-L-methionine (SAM or AdoMet) radical enzyme that catalyzes reduction ofthe AdoMet sulfonium to produce 5′-deoxyadenosyl radicals, high-energy carbon radicals that can directlyabstract hydrogen atoms from dethiobiotin. The available experimental and structural data suggest that a[2Fe–2S]2+ cluster bound deep within biotin synthase provides a sulfur atom that is added to dethiobiotinin a stepwise reaction, first at the C9 position to generate 9-mercaptodethiobiotin, and then at the C6 positionto close the thiophane ring. The formation of sulfur-containing biomolecules through a radical reaction in-volving an iron–sulfur cluster is an unprecedented reaction in biochemistry; however, recent enzyme discov-eries suggest that radical sulfur insertion reactions may be a distinct subgroup within the burgeoning RadicalSAM superfamily. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Although sulfur-containing biomolecules are found in all knownorganisms, they represent just a small fraction of the total biochem-ical repertoire. Beyond the amino acids cysteine and methionine,abundant organosulfur compounds include glutathione, S-adenosyl-L-methionine, S-adenosyl-L-homocysteine, 5′-methylthioadenosine, andL-homocysteine. In addition, most organisms require several sulfur-containing coenzymes and cofactors, including coenzyme A, lipoic acid,biotin, thiamine, and molybdopterin. Microorganisms and plants areknown to generate numerous sulfur-containing natural productssuch as mycothiol (M. tuberculosis), penicillin (Pennicillium fungi),or allicin (A. sativum or garlic). More recently, several sulfur contain-ing post-translationally modified nucleotides and amino acids havebeen discovered, including for example, 2-thiouridine, 2-methylthio-N6-isopentenyladenosine, and 3-methylthioaspartate.

, S-adenosyl-L-homocysteine;xononanoate; CoA, coenzymeelargonic acid; DTB, dethiobio-DOR, electron-nuclear double, electron spin echo envelope

-methylthioadenosine/AdoHcy/

cal SAM enzymes and Radical

ger 245, Honolulu, HI 96822-08.

rights reserved.

Themajority ofwell-characterized biosynthetic pathways for sulfur-containing biomolecules involve polar mechanisms that take advan-tage of the reactivity of sulfur as a nucleophile and leaving group. Forexample, the pathway for thiamine biosynthesis involves a sulfuratom from cysteine that is transferred first to a peptide to generatea thiocarboxylate, and then to an enzyme-bound 1-deoxyxylulose-5-phosphate imine, with the final sulfur insertion proposed tooccur through nucleophilic carbonyl addition and acyl substitutionreactions [1]. However, several biosynthetic pathways are now knownto involve substitution of sulfur for hydrogen at unactivated aliphaticor electron-rich aromatic carbon atoms; these reactions almost certain-ly proceed by mechanisms that avoid nucleophilic sulfur compounds[2]. For example, the thiophane ring of biotin is formed when dethio-biotin (DTB, 5-methyl-2-oxo-4-imidazolidinehexanoic acid) reactswith the iron–sulfur-cluster-containing AdoMet radical enzyme bio-tin synthase, with the sulfur atom derived from within the purifiedenzyme; this reaction requires the substitution of sulfur for hydro-gen at two unactivated aliphatic carbon positions [3,4]. Common toall of these mechanistically difficult sulfur insertion reactions is theinvolvement of an AdoMet radical enzyme that contains at leasttwo iron–sulfur clusters. This review focuses on the dual role ofiron–sulfur clusters in biotin synthase as both the substrate and thecatalytic cofactor for the sulfur insertion reaction.

2. Why does biotin contain a sulfur atom?

Biotin is an enzyme cofactor that is used in carboxylation, decar-boxylation, and transcarboxylation reactions in most organisms. In a

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typical biotin-dependent carboxylase [5], bicarbonate is activated anddehydrated through a reaction with ATP, and the resulting carbondioxide is captured as a carbamate derivative with N1′ from theimidazolidone ring [6]. The carbon dioxide is typically released ina second active site in the presence of carbon nucleophiles such asthe enolate of acetyl CoA or pyruvate, generating carboxylatedproducts such as malonyl CoA or oxaloacetate. In principle, thistype of step-wise carboxylation reaction could also be accomplishedby the biotin precursor DTB (as well as by several amino acid resi-dues), which might lead one to question why evolution mightfavor extension of the biosynthetic pathway solely for the additionof a single sulfur atom.

Chemically, biotin consists of the catalytic 2-imidazolidone ringfused to a five-membered thiophane ring (aka. tetrahydrothiophene)and a pentanoic acid tail, with the latter providing a means forcovalently attaching biotin to a lysine residue on biotin-dependentenzymes. Although the thiophane ring does not play a direct rolein catalysis, the cis chirality at the ring fusion interface places thesulfur atom 3.22 Å from the N1′ position of the imidazolidone ringwhen the thiophane ring is in the more stable endo conformation(Fig. 1, left panel) [7]. Several studies have examinedwhether the sulfuratom could affect the reactivity of theN1′ position through a transannu-lar electronic effect, but most have concluded that the effect is eitherexceedingly small or nonexistent [7–9]. Perhaps the only plausibleexplanation for the presence of sulfur in biotin comes from a com-parison by Tipton and Cleland of the stability of N1′-carboxybiotin[10] and N-carboxy-2-imidazolidone [11], which suggests that thesteric bulk of the thiophane ring (Fig. 1, right panel) may be importantfor minimizing acid-catalyzed spontaneous decarboxylation, therebyincreasing the efficiency of the enzymatic carboxylation reaction. Sulfurincorporation to form the thiophane ring is the last step in the biotinbiosynthetic pathway, and this step presents a unique chemical chal-lenge to the enzyme that catalyzes this transformation.

3. Biotin biosynthesis

3.1. The biotin biosynthetic pathway

The current view of the biotin biosynthetic pathway is summa-rized in Fig. 2. Two biosynthetic methods for generating the commonprecursor, pimeloyl ACP, have been characterized. Cronan and co-workers have recently presented evidence that in E. coli, BioC is amethyltransferase that caps the terminal carboxylate group of malo-nyl CoA, which then serves as a substrate for 3 rounds of fatty acidbiosynthesis [12]. At this point, BioH hydrolyzes the methyl ester togenerate pimeloyl ACP [12]. An alternate pathway for generatingpimeloyl ACP has been characterized in B. subtilis. The cytochromeP450 enzyme BioI binds long-chain fatty acyl ACP derivatives, andcatalyzes successive rounds of hydroxylation at the C7–C8 positions

Fig. 1. The three-dimensional structure of carboxybiotin. (left) N1′-carboxybiotin consists oenvelope conformation with the sulfur atom 0.84 Å [7] or ~22° [87] out of the thiophane plathe thiophane ring, as can be seen in this space-filling model using average van der Waal radred; sulfur, yellow. Adapted from a structure of the streptavidin:biotin complex (PDB ID: 3

to produce pimeloyl ACP [13]. A genomic analysis suggests that addi-tional complementary pathways may exist for the biosynthesis ofpimeloyl CoA or pimeloyl ACP in other organisms [14]. In some organ-isms, pimeloyl CoA can also be formed from free pimelic acid throughthe action of BioW, an ATP-dependent acyl CoA synthetase [15]. Cro-nan and coworkers have suggested that pimeloyl ACP may be theendogenous substrate for the next biosynthetic step, although invitro studies indicate that pimeloyl CoA also serves as an efficientsubstrate [12]. Although further in vitro studies are clearly needed,in some organisms BioF could have evolved the ability to use eitherpimeloyl ACP or pimeloyl CoA to facilitate utilization of either en-dogenous pimeloyl ACP or exogenous scavenged pimelic acid.

The remaining four steps are conserved in all organisms thatbiosynthesize biotin (Fig. 2). 8-Amino-7-oxononanoate (AON) synthase(BioF) is a PLP-dependent α-oxoamine synthase that catalyzes con-densation of L-alanine with pimeloyl CoA, followed by decarboxyl-ation to generate AON, CO2, and CoA [16]. 7,8-Diaminopelargonicacid (DAPA) synthase is PLP-dependent aminotransferase that transferstheα-amino group from S-adenosyl-L-methionine to the C7 position ofAON, generating DAPA and S-adenosyl-4-methylthio-2-oxobutanoate[17]. Dethiobiotin synthetase (BioD) is an ATP-dependent kinase thatcatalyzes phosphorylation of N7-carboxy-DAPA and the subsequentring closure, generating the intact 2-imidazolidone ring of DTB togetherwith ADP and Pi [18]. Biotin synthase (BioB) uses two equivalents ofAdoMet to remove two hydrogen atoms from DTB, generating 5′-deoxyadenosine and methionine, and incorporates sulfur from aniron–sulfur cluster to generate the biotin thiophane ring.

3.2. Evidence that DTB is the true precursor to biotin

In order to accurately establish the chemical mechanism of biotinsynthase, the true in vivo substrate must first be identified. Severalearly mechanistic proposals centered on the possibility of a previous-ly undetected biotin precursor with a hydroxyl or sulfhydryl group atthe C6 and/or C9 positions; for example, Lezius et al. [19] proposedthat biotin was formed through the condensation of pimeloyl CoAand N-carbamoyl-L-cysteine. However, feeding studies were moreconsistent with DTB as a precursor to biotin. DTB is readily producedby Raney nickel reduction of biotin [20], and this chemical productwas shown to be nearly as effective as biotin in supporting the growthof S. cerevisiae [21], Neurospora [22], and Lactobacillus arabinosus [23].Direct evidence for the in vivo conversion of DTB to biotinwas provid-ed by Tepper et al. [24], who showed that Aspergillus niger will convert(14C-carbonyl)-DTB into (14C-carbonyl)-biotin. Evidence that DTBwas utilized in the step catalyzed by BioB was provided throughcross-feeding studies with a complementary set of E. coli K12 biotinauxotrophs [25–27]. One mutant was noted that produced DTB intothe extracellular media [28], and a subset of biotin auxotrophs thatgrow on biotin but not DTB (i.e., bioB mutants) were able convert

f a planar imidazolidone ring fused to a thiophane ring, existing primarily in the endone. (right) The carboxy group on N1′ of biotin is partially protected by the steric bulk ofii for each atom. Color scheme: carbon, cyan; hydrogen, white; nitrogen, blue; oxygen,RY2).

Fig. 2. The complete biotin biosynthetic pathway in E. coli and B. subtilis. In E. coli, pimeloyl ACP is synthesized from malonyl CoA, using the methyltransferase BioC and the esteraseBioH to temporarily mask and unmask the terminal carboxylate, thereby allowing the growing chain to pass three times through the fatty acid biosynthetic pathway. In B. subtilis,long chain fatty acyl ACPs are oxidized at the C7 position by the cytochrome P450 BioI, generating pimeloyl ACP. The reminder of the biosynthetic pathway is conserved among allorganisms, utilizing BioF, BioA, and BioD to incorporate L-alanine, an amino group derived from AdoMet, and the imidazolidone carbonyl derived from carbon dioxide. DTB is thenconverted to biotin by insertion of a sulfur atom as catalyzed by the AdoMet radical enzyme BioB (blue boxed region). At each step, the newly added or transformed portion of themolecule is highlighted in red.

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DAPA to DTB in cell-free extracts [29,30]. Together, these studies pro-vide strong evidence that DTB is an intermediate in the biotin biosyn-thetic pathway, but do not necessarily demonstrate that DTB is thesubstrate for biotin synthase, since they could not exclude the possi-ble involvement of an additional undetected enzyme.

Since a reaction that would result in conversion of DTB to biotinwas biochemically unprecedented, several studies examined whetherthe process might involve prior introduction of a functional group atC6 or C9 by an unidentified enzyme. Both C6 and C9 unsaturatedalkenes [31] and hydroxylated analogs [32] of DTB were excluded asprecursors as these could not support growth of an E. coli biotin auxo-troph. Parry and coworkers examined whether tritium incorporatedregiospecifically at C5 through C9 in DTB was retained in biotinfollowing conversion in A. niger, and concluded that the C5, C7, andC8 hydrogen atoms were unaffected, excluding the possibility of analkene intermediate in between DTB and biotin in the pathway [33].Marquet and coworkers examined whether a C6 and/or C9 thiolatedanalog of DTB could be an intermediate. In E. coli, they found thatboth 35S-labeled 6- and 9-mercaptodethiobiotin were able to supportgrowth and were converted into biotin, however this occurred withloss of the labeled sulfur. Thus the results of in vivo studies were atbest ambiguous: while DTB is clearly a viable biotin precursor and isproduced by bioB mutants, thiolated biotin precursors could not beexcluded. Further, the in vivo source of the sulfur atom in biotincould not be established with any certainty [34,35]. Further progresswould require establishing a defined in vitro enzymatic assay.

4. Biotin synthase

4.1. Establishing the components of a defined in vitro assay

While the production of biotin could be detected in bacteria andfungi, cell-free systems remained unable to produce any biotin, which

made mechanistic studies difficult and ambiguous. A major break-through came with the identification and sequencing of the E. colibio operon [36], which facilitated the production of cell free extractscontaining a significant amount of recombinant biotin synthase.Ifuku et al. examined whether various small biomolecules andmetal ions stimulated the production of biotin from DTB in cell-freeextracts of E. coli containing recombinant biotin synthase, andnoted an ~20-fold increase when Fe2+, KCl, NADPH, and AdoMetwere added [37]. Flint and coworkers described the first purifica-tion of E. coli biotin synthase and found that ~1 turnover perhour was observed when the assay contained DTB, AdoMet,NADPH, Fe2+, and a crude cell-free extract from a Δbio E. colistrain [38]. The requirement for a crude extract suggested a miss-ing protein component with a gene outside of the bio operon.The missing components in the crude extract were subsequentlyidentified as flavodoxin I (FldA) and ferredoxin(flavodoxin):NADP+ oxidoreductase (Fpr) [39,40]. Around this time, severalgroups noted the similarity between the components requiredfor biotin synthase activity and those required for the AdoMetradical enzymes pyruvate formate-lyase activating enzyme, classIII ribonucleotide reductase, and lysine-2,3-aminomutase[38–41]. Since these enzymes were known to contain essentialair-sensitive iron–sulfur clusters, we added dithiothreitol and sulfideto in vitro assays to facilitate in situ chemical reconstitution of FeS clus-ters [42] resulting in a further improvement in activity.

Several other factors have exhibited a stimulating effect in variousearly studies, but have not shown a reproducible effect with purifiedbiotin synthase, including L-cysteine, fructose 1,6-bisphosphate,thiamine pyrophosphate, and MioC. While there is little evidencefor the direct involvement of these additives in the production ofbiotin, the stimulating effect may result from these additives react-ing with unidentified components of crude extracts or partially puri-fied enzyme preparations to create assay conditions favorable to

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biotin production. When 35S-cysteine was incubated with crude ex-tracts containing biotin synthase, biotin was produced that was~80% labeled with 35S [34,43], although no incorporation wasobserved with purified enzyme [44]. It was later proposed that biotinsynthase might have an intrinsic PLP-dependent cysteine desulfuraseactivity [45]. However, a PLP-deficient E. coli strain (ΔpdxH) wasshown to be competent for the in vivo conversion of dethiobiotin to bi-otin [46], and other studies could not reproduce the desulfurase activity[47,48]. An alternative explanation for the apparent effect of L-cysteinein crude extracts and partially purified BS could be that a trace amountof a contaminating cysteine desulfurase (IscS, SufSE, or similar) couldhave resulted in the production of sulfide from cysteine, leading tomore complete FeS cluster reconstitution. Fructose 1,6-bisphosphatewas found to be stimulating in cell-free extracts of E. coli while otherglycolysis metabolites were found to have no effect or showed only amodest increase in biotin production [37]. The excess fructose 1,6-bisphosphate may have channeled through glucose 6-phosphate intothe pentose phosphate pathway, resulting in the recycling of NADPHand a lower overall redox potential for the biotin synthase assay.Similarly, addition of thiamine pyrophosphate to crude extracts[40] may have provided some additional reducing power, as it is acofactor required by pyruvate ferredoxin oxidoreductase, whichreduces ferredoxin or flavodoxin in the process of converting pyruvateto acetyl CoA [49]. MioC is a flavoprotein [50] that has been structurallyidentified as a member of the flavodoxin superfamily [51], and while itis not essential for biotin synthase activity, it may serve to augment theotherwise dismal kinetic activity of the E. coli flavodoxin I/Fpr reducingsystem [52].

4.2. Biotin synthase is an AdoMet radical enzyme

Initially, the discovery that AdoMet enhances the production ofbiotin in cell-free extracts led to the suggestion that it might be thesulfur donor for the biotin thiophane ring [37]. However, Florentinet al. [41] found that when 35S-AdoMet was added to cell-freeextracts of B. sphaericus, the radiolabel was not incorporated into bi-otin. Further, 35S-AdoMet did not serve as the sulfur donor for purifiedE. coli biotin synthase [44]. The requirement for AdoMet, NADPH, andflavodoxin I led several authors to propose that biotin synthase couldbe an AdoMet radical enzyme [38–41]. Marquet and coworkers dem-onstrated that the conversion of DTB to biotin is accompanied by thereductive cleavage of AdoMet to 5′-deoxyadenosine (5′-dAH) andmethionine, a reaction now known to be characteristic of the RadicalSAM superfamily [53]. Shaw and coworkers confirmed this resultand reported an approximate 2:1 ratio of methionine to biotin pro-duction [54]. Marquet and coworkers then used chemically deuterat-ed DTB and mass spectrometry to demonstrate that the 5′-dAHproduced incorporated deuterium from both the C6 and C9 positionsof DTB, suggesting that both positions are activated via hydrogenatom abstraction by a 5′-deoxyadenosyl radical [55]. Finally, biotinsynthase contains a characteristic cysteine motif, C53xxxC57xxC60,that binds a catalytic [4Fe–4S] cluster in members of the RadicalSAM superfamily [56]. Mutagenesis of any of these Cys residues toSer [57] or Ala [58] abolishes all biotin production, consistent witha catalytic role for the associated iron–sulfur cluster.

4.3. Biotin synthase contains both [2Fe–2S]2+ and [4Fe–4S]2+/+ clusterbinding sites

When biotin synthase is purified aerobically, the protein is a homodi-mer that contains one air-stable [2Fe–2S]2+ cluster per monomer [38].Since AdoMet radical enzymes are known to require a [4Fe–4S]2+/+

cluster, numerous studies have examined the properties of endogenousand reconstituted clusters in an attempt to identify the active form ofthe enzyme and to understand the role of FeS clusters in catalysis [59].Johnson and coworkers demonstrated that biotin synthase containing

a [2Fe–2S]2+ cluster could be chemically reduced and transformedinto a protein containing a substoichiometric amount of a [4Fe–4S]2+

or [4Fe–4S]+ cluster [60]. Exposure of this cluster to oxygen results ina partial conversion back to a [2Fe–2S]2+ cluster [47,61]. Usingmild re-constitution procedures and spectroelectrochemistry, Ugulava et al.demonstrated that the [4Fe–4S]2+ cluster could be assembled and re-duced either in the presence or absence of the [2Fe–2S]2+ cluster[62,63]. Using Mössbauer spectroscopy of biotin synthase in whicheach of these two cluster types were differentially labeled with 57Fe,Ugulava et al. further demonstrated that the [2Fe–2S]2+ and [4Fe–4S]2+ cluster occupy different sites within the protein, and proposedthat three additional highly conserved Cys residues, Cys97, Cys128,and Cys188, serve as ligands to the [2Fe–2S]2+ cluster [64]. Biotinsynthase reconstituted to contain both the [2Fe–2S]2+ and [4Fe–4S]2+

clusters produced nearly stoichiometric amounts of biotin in the absenceof additional Fe2+/3+ and/or S2− [42].

4.4. The structure of E. coli biotin synthase

E. coli biotin synthase is a homodimer that is purified with one[2Fe–2S]2+ cluster per 38 kD monomer [38]. Under anaerobic condi-tions, mild reconstitution can produce an additional [4Fe–4S]2+ clus-ter per monomer [63], which is stabilized by the binding of AdoMetand dethiobiotin [65]. This reconstituted form was crystallized andthe structure solved to 3.4 Å resolution using X-ray diffraction [66].Each monomer is an (αβ)8 barrel (TIM barrel) encapsulating the sub-strates and iron–sulfur clusters within the core of the barrel (Fig. 3A),with the [4Fe–4S]2+ cluster positioned closest to the protein surfaceto facilitate electron transfer from reduced flavodoxin. The [4Fe–4S]2+

cluster is coordinated by the three conserved cysteine residues(Cys53, Cys57, and Cys60) found within an extended loop that con-nects β-strand 1 and helix 1 (Fig. 3B). A unique iron of the [4Fe–4S]2+

cluster is coordinated by the methionyl amine and carboxylate groupsof AdoMet, positioning the sulfonium of AdoMet ~4.0 Å from the cluster.AdoMet is also held in place by hydrogen bonds between the riboseand Asp155 and Asn153, as well as between the carboxylate andArg173. The adenine sits in a hydrophobic pocket generated by Tyr59,Ile192, and the carboxylate chain of DTB. Buried deep within the activesite, the [2Fe–2S]2+ cluster is coordinated by Cys97, Cys128, Cys188,and Arg260 (Fig. 3C). DTB is sandwich between AdoMet and the [2Fe–2S]2+ cluster and is held in place by hydrogen bonds between theimidazolidone ring and Asn151, Ans153, and Asn222. The C9 positionof DTB is ~3.9 Å from the C5′ position of AdoMet, consistent with directhydrogen atom abstraction by a transient 5′-deoxyadenosyl radicalfrom this position in the substrate. The C9 position of DTB is also~4.6 Å from the μ-sulfide of the [2Fe–2S]2+ cluster. This FeS clusterand the associated cysteine residues contain the only sulfur atomswith-in the active site, and presumably one of these sulfur atoms must bedestined for incorporation into the biotin thiophane ring.

5. The mechanism of sulfur insertion catalyzed by biotin synthase

5.1. The properties of the iron–sulfur clusters

The two different iron–sulfur clusters in biotin synthase have uniquechemical and physical properties presumably adapted to their function.The [2Fe–2S]2+ cluster is air-stable and has distinctive UV/visibleabsorption bands at 332 and 452 nm (Fig. 4A) that give rise to thecharacteristic reddish-brown color of the aerobically purified enzyme[38]. The [2Fe–2S]2+ cluster has a distinctive narrow Mössbauer quad-rupole doublet (δ=0.29 mm/s, ΔEQ=0.53 mm/s [47,64,67]) and givesrise to strong vibrational bands at 301, 331, and 349 cm−1 observableby resonance Raman spectroscopy [47,60]. This cluster dissociatesfrom the protein when reduced with strong chemical reductants suchas sodiumdithionite [62], but is not reduced by flavodoxin. For this rea-son, biotin synthase is one of the few AdoMet radical enzymes that is

Fig. 3. The structure of biotin synthase from Escherichia coli. (A) Biotin synthase is a homodimer, with each 38.6 kD monomer forming an (αβ)8 barrel that encapsulates the activesite. The [4Fe–4S]2+ cluster is bound near the surface of the enzyme, while the [2Fe–2S]2+ cluster is bound deep within the barrel (Fe, orange; S, yellow), and AdoMet (magenta)and DTB (cyan) are sandwiched in between. (B) Two views of AdoMet coordinated via the methionyl amine and carboxylate to a unique Fe within the [4Fe–4S]2+ cluster. The riboseis held in position by hydrogen bonds to Asn153 and Asp155, while the adenine ring lies in a pocket formed by Tyr59, Ile192, and DTB. The sulfonium is positioned ~4.0 Å from theFeS cluster. (C) DTB is held in between AdoMet and the [2Fe–2S]2+ cluster by hydrogen bonds between the imidazolidone ring and Asn151, Asn153, and Asn222. Structure fromadapted from Berkovitch et al. [66] (PDB ID: 1R30).

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Fig. 4. Spectra of the [2Fe–2S] and [4Fe–4S] clusters. (A) UV/visible spectra of the[2Fe–2S]2+ cluster (solid curve) with peaks at 332 and 455 nm, and the [4Fe–4S]2+

cluster (dashed curve) with a broad peak at ~400–420 nm. (B) EPR spectra of the[2Fe–2S]+ cluster intermediate (solid curve), proposed to incorporate 9-MDTB as abridging thiolate ligand, and the [4Fe–4S]+ cluster (dashed curve), formed followingelectron transfer from flavodoxin but prior to reductive cleavage of AdoMet.

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not significantly active in the presence of dithionite. The reduced[2Fe–2S]+ cluster has been observed in two inactive mutant en-zymes, Asn153Ala and Asp155Ala, and exhibits a narrow axial EPRsignal at g=2.00, 1.91 [68]. A different EPR signal associated withthis cluster has been observed during catalysis (discussed below). Inthe absence of turnover, the various spectroscopic signals associatedwith the [2Fe–2S]2+ cluster do not appear to be significantly affectedby the presence or absence of DTB or AdoMet.

In contrast, the [4Fe–4S]2+ cluster is lost from the protein withinminutes of exposure to air [47,65] and is not present in the aerobicallypurified enzyme [60]. This cluster can be reconstituted with a stoi-chiometric amount of Fe3+, S2−, and excess dithiothreitol in aqueousbuffer [63], or with Fe3+, S2−, and dithionite in 60% ethylene glycol[60,63]. The [4Fe–4S]2+ cluster has a broad UV/visible absorptionband centered at ~410 nm (Fig. 4A) that gives rise to a faint yellowcolor [60,62], a characteristic broad Mössbauer quadrupole doublet(two overlapping doublets at δ=0.44, 0.45 mm/s, ΔEQ=1.28,1.03 mm/s [47]), and exhibits a vibrational band at 338 cm−1

observable by resonance Raman spectroscopy [47,60]. The [4Fe–4S]2+

cluster is reduced by dithionite with Em≈−430–450 mV [63] and pro-duces a [4Fe–4S]+ cluster with an axial EPR signal typical of iron–sulfurclusters with g=1.94, 2.04 (Fig. 4B) [60–62].

5.2. Roles for the iron–sulfur clusters

The crystal structure of biotin synthase shows an intimate associ-ation between the [4Fe–4S]2+ cluster and AdoMet, with direct coor-dination of the AdoMet amine and carboxylate to the cluster. Similarcoordination had previously been demonstrated in pyruvateformate-lyase activating enzyme, using ENDOR spectroscopy of

15N, 17O, and 13C labeled AdoMet bound to protein containing a[4Fe–4S]+ cluster [69,70]. In biotin synthase, shifts in resonanceRaman vibrational bands, Mössbauer quadrupole doublets, and EPRg values all suggested a similar coordination of AdoMet to the [4Fe–4S]2+ cluster [71]. The effect of this association is to bring the[4Fe–4S]+ cluster into close contact with the AdoMet sulfonium.Hoffman and coworkers have suggested that partial isotropic cou-pling between the electron spin in the reduced cluster and the nucle-ar spin in (13C-methyl)-AdoMet indicates partial orbital overlapbetween these species [69]. Recent QM/MM calculations of the[4Fe–4S]+ cluster/AdoMet complex in biotin synthase predict thatthe highest energy ground-state molecular orbital (SOMO) is largelylocalized to the [4Fe–4S]+ cluster, but the lowest energy excited-state molecular orbital (LUMO) is extensively delocalized betweenthe FeS cluster and the sulfonium C5′-S σ⁎ orbital [72]. Thus the pre-dicted role of [4Fe–4S]2+ cluster is to transfer an electron into AdoMet,generating an unstable excited state that relaxes through reductivecleavage of the sulfonium and generation of a 5′-deoxyadenosyl radical.The cluster may also help select the correct C\S bond for cleavage byoverlap between the FeS cluster orbitals and the AdoMet sulfoniumC5′-S σ⁎ orbital.

Early in vitro assayswith purified biotin synthase had suggested thatthe sulfur donor was present in the enzyme following purification[44,73], indicating that the sulfur donorwas either the [2Fe–2S]2+ clus-ter, a protein residue, or a tightly-bound molecule. Marquet and co-workers reconstituted unlabeled apoenzyme with Fe3+, 34S2−, andDTT and assayed for conversion of DTB to biotin in the presence ofunlabeled AdoMet. Although the amount of biotin produced wasvery low, the biotin incorporated ~65% 34S label [74]. A more recent in-vestigation substituted selenide for sulfide in the reconstitution proce-dure. When biotin synthase containing only Se2− was assay in thepresence of S2− in the buffer, ~0.3–0.9 equiv. of biotin was producedcontaining 46–72% Se2− [75]. UV/visible [42] and Mössbauer [67,76]studies are consistent with consumption or destruction of the [2Fe–2S]2+ cluster during biotin production. For example, reconstituted bio-tin synthase is reported to contain ~30% of the total Fe in [2Fe–2S]2+

clusters prior to an assay, which is reduced to ~15% following incuba-tion under assay conditions for 4 h [67]. Curiously, this decrease in[2Fe–2S]2+ cluster content does not appear to correlate with the rateof biotin production [76]. However, although the precise mechanisticand kinetic details are somewhat uncertain, the predicted role of the[2Fe–2S]2+ cluster is to provide a sulfur for incorporation into the biotinthiophane ring.

5.3. The formation of 9-mercaptodethiobiotin as a catalytic intermediate

Conversion of DTB to biotin requires the abstraction of two hydro-gen atoms from the C6 and C9 positions [55]. Early studies had indi-cated that the reaction requires two equiv. of AdoMet [54], althoughclearly only 1 equiv. of AdoMet can bind to each active site [66]. Thissuggests that an intermediate must exist that remains bound tothe enzyme while 5′-dAH and methionine dissociate and a secondequivalent of AdoMet binds. Shaw and coworkers used 14C-DTBand detected a compound that was produced within 30 min fromDTB, and when purified by HPLC can then be converted to 14C-biotinby biotin synthase [54]. However, they were not able to chemicallyidentify this intermediate. Marquet and coworkers chemically syn-thesized 9-mercaptodethiobiotin (9-MDTB), and showed that biotinsynthase can transform this putative intermediate into biotin at arate comparable to the rate of turnover with DTB, albeit with avery high apparent Km≈200 μM (compared with Km=5 μM forDTB) [48]. Taylor et al. used HPLC mass spectrometry to search forany new species formed during an assay with a mass correspondinga monothiolated intermediate, and detected a minor species thatelutes after DTB on reversed-phase HPLC and was formed at a con-centration corresponding to ~0.2–0.3 equiv. per biotin synthase

1219C.J. Fugate, J.T. Jarrett / Biochimica et Biophysica Acta 1824 (2012) 1213–1222

dimer [77]. Using (2H3-9-methyl)-DTB, Taylor demonstrated thatthis new molecule is monothiolated specifically at C9, and thusmost likely corresponds to 9-MDTB. Interestingly, 9-MDTB is formedas the sole product of turnover in several mutant enzymes that werepreviously thought to be inactive, such as Asn153Ser biotin synthase[77,78]. With the WT enzyme and unlabeled DTB, 9-MDTB is formedin a relatively rapid burst within ~5 min following initiation of theassay by addition of DTB (k1≈0.1 min−1), and then decays to formbiotin (k2≈0.4 min−1). The concentration of 9-MDTB reaches anapparent steady-state concentration of ~0.1 equiv. per BS dimerafter 1 h [77,79].

5.4. The formation of a paramagnetic [2Fe–2S]+ cluster as a catalyticintermediate

If a sample of biotin synthase is frozen during turnover, a complexmixture of paramagnetic species is observed by EPR spectroscopy[42,76,80]. In addition to a sharp isotropic signal due to the flavodoxinFMN semiquinone, two overlapping rhombic signals are observed inan approximate 2:1 ratio with g=1.880, 1.955, 2.010 and g=1.845,1.940, 2.000 (Fig. 4B) [76,80]. Hyunh and coworkers observed the for-mation ~0.3 spins per BS monomer within 15 min of initiating anassay, and note that in parallel 57Fe-labeled samples, the Mössbauerspectrum shows development of a minor component attributable toa [2Fe–2S]+ cluster [76]. Taylor et al. showed that mutagenesis ofthe [2Fe–2S] cluster ligand Arg260 to Met results in a consolidationof the two paramagnetic signals into a single rhombic signal with in-termediate g values, and also results in the loss of a triplet signal inESEEM spectra due to coupling of the electron spin to 14N from theArg260 guanidino group [80]. Together, these results confirm thatthe unpaired electron spin resides in the [2Fe–2S]+ cluster. Tayloret al. also observed that the formation of this reduced FeS clustercorrelates with the production of 9-MDTB (k≈0.1 min−1), whilebiotin production lags behind by ~20 min at 25 °C [80]. Thus thereduced [2Fe–2S]+ cluster appears to be a catalytic intermediateassociated with formation of the monothiolated intermediate9-MDTB.

5.5. Evidence that biotin synthase is half-site active and undergoes slowsteady-state turnover

In vitro biotin synthase assays have been plagued by exceeding-ly slow turnover and low yields of biotin. Ironically, biotin produc-tion is slightly improved in the presence of cell-free bacterialprotein extracts [44], suggesting that an additional protein compo-nent, while not essential, is somehow assisting the reaction. Severalrecent studies have since demonstrated that biotin synthase is sub-ject to cooperative product inhibition by 5′-dAH and methionine[79,81,82], such that in typical assays after one turnover the en-zyme would be ~50% inhibited [79]. This product inhibition isrelieved by the action of 5′-methylthioadenosine/AdoHcy/5′-dAHnucleosidase (MTAN) [78,81,83], which efficiently cleaves 5′-dAH(Km=4.6 μM, kcat=5.1 s−1); this enzyme would be present in crudebacterial protein extracts but is absent from purified biotin synthase.

Biotin synthase is also strongly inhibited by AdoHcy (Ki≤650 nM)[79]. Although AdoHcy is not a product of the biotin synthase reac-tion, it is present both in vivo and as a common contaminant in com-mercial AdoMet preparations, and most prior studies probably wereimpaired by a significant amount of AdoHcy inhibition. Further, thecompetitive inhibition patterns obtained when the concentration ofAdoHcy and AdoMet in an assay were systematically varied showedclear signs of cooperative behavior; the data could only be modeledby assuming that BS dimer containing one AdoMet and one AdoHcyin opposing monomers was catalytically inactive [79]. In assays thatcontained 25 μM biotin synthase monomer (12.5 μM dimer), only10.6 μM AdoHcy was needed to abolish activity, suggesting that

there are only ~0.85 actives sites per BS dimer [79]. Similar resultswere also obtained with the natural product sinefungin (Ki=75 μM)[79]. Curiously, equilibrium binding studies showed that two equiva-lents of AdoMet and DTB bind to each BS dimer, with no evidenceof cooperative behavior [65,79]. Together, these inhibition studieshint at strong negative cooperativity within the biotin synthasedimer that affects catalysis (kcat) but not binding substrate or inhib-itor binding (Km or Kd).

In the presence of MTAN to relieve inhibition by AdoHcy and 5′-dAH, and using highly purified biosynthetic AdoMet, biotin synthaseexhibits a burst of biotin production within the first 5–15 min, fol-lowed by very slow steady-state turnover for at least 4 h [79]. Themagnitude of the burst is equal to the concentration of biotin synthasedimer, again suggesting that only one active site per dimer is activeat any one time; i.e. that the enzyme is half-site active. When the[2Fe–2S]2+ cluster contains 32S2−, while the [4Fe–4S]2+ cluster andthe buffer contain exclusively 34S2−, the biotin produced in the burstcontains only 32S [79]. All subsequent turnovers of biotin productioncontain 34S, presumably from the buffer, suggesting either that the[2Fe–2S]2+ cluster is being slowly reassembled in situ with S2− fromthe buffer, or that S2− alone is capable of serving as a less efficientalternate substrate in subsequent turnovers. Distinguishing betweenthese alternative scenarioswill require developingmethods to track thefate of sulfide and the formation of iron–sulfur clusters on the enzymeduring catalysis.

5.6. A proposed mechanism for biotin thiophane ring formation

A mechanism that describes the first turnover of biotin formationis depicted in Fig. 5. Electron transfer from flavodoxin into biotinsynthase results in reduction of the [4Fe–4S]2+ cluster. An electronin the [4Fe–4S]+ cluster is excited into the LUMO, a mixed molecu-lar orbital that includes the AdoMet C5′-S σ⁎ orbital, and the bondrapidly cleaves to generate a 5′-deoxyadenosyl radical. This radicalabstracts a hydrogen atom from the C9 methyl group of DTB, gener-ating a dethiobiotinyl carbon radical. This radical is quenched byreacting with the nearby μ-sulfide of the [2Fe–2S]2+ cluster, andin the process an electron is transferred from the sulfide into theFeS cluster, reducing this cluster to a [2Fe–2S]+ cluster. The precisenature of the resulting enzyme intermediate is unclear; our currentproposal is that 9-MDTB remains as a symmetric bridging thiolateligand to the [2Fe–2S]+ cluster, which would require a significantstructural shift in the DTB/9-MDTB binding site. At this point, 5′-dAH and methionine dissociate and a second equivalent of AdoMetbinds. The second half-reaction can proceed in a manner identicalto the first half-reaction, except now directed at the C6 pro-S hydro-gen atom. Formation of the second C\S bond is again accompaniedby the transfer of an electron into the FeS cluster. The resulting difer-rous cluster is unstable and dissociates from the enzyme, along with asecond equivalent of 5′-dAH and methionine and the ultimate prize,biotin.

6. Insights into radical C\S bond formation:Why use an iron–sulfurcluster as sulfur donor?

Themajority of biosynthetic reactions involving sulfur take advan-tage of the nucleophilic character of thiolates and thiocarboxylatesand follow polar mechanisms. A polar mechanism requires that thebiosynthetic precursor have an electrophilic carbon atom at the de-sired site of C\S bond formation. However, some sulfur-containingbiomolecules are generated from nonpolar or electron rich precursorssuch as an octanoyl-protein (lipoic acid precursor) or adenosine (2-methylthio-N6-isopentenyladenosine precursor). Unfortunately, evo-lution of the biotin biosynthetic pathway resulted in a precursor,dethiobiotin, that has nonpolar unactivated carbon atoms at thedesired sites of C\S bond formation. In answer to this apparent

Fig. 5. A proposed mechanism for the formation of the biotin thiophane ring. (A) Electron transfer into the [4Fe–4S]2+ cluster results in population of an excited state that includesthe AdoMet C5′-S σ* orbital, resulting in spontaneous C\S bond cleavage and generation of a 5′-deoxyadenosyl radical. (B) Abstraction of a hydrogen atom from the C9 position ofdethiobiotin generates a dethiobiotinyl C9 carbon radical, which is quenched by a μ-sulfide from the [2Fe–2S]2+ cluster. Inner sphere electron transfer from the sulfide into the FeScluster results in reduction to a [2Fe–2S]+ cluster. Following exchange of 5′-dAH and methionine for a second equiv. of AdoMet and reductive cleavage to generate another 5′-deoxyadenosyl radical, abstraction of a hydrogen atom from the C6 position of 9-MDTB generates a C6 carbon radical, which is quenched by the bridging thiolate ligand togenerate the thiophane ring of biotin. Transfer of an additional electron from the thiolate into the FeS cluster results in an unstable diferrous cluster that most likely dissociatesfrom the protein.

1220 C.J. Fugate, J.T. Jarrett / Biochimica et Biophysica Acta 1824 (2012) 1213–1222

chemical dilemma, AdoMet radical enzymes can activate aliphatic C\Hbonds by hydrogen atom abstraction, resulting in transient formation ofa highly reactive substrate-centered carbon radical that can then befunctionalized by various heteroatoms. However, the use of radicalmechanisms brings along a whole new set of problems.

Carbon radicals are notoriously reactive toward hydrogen atomabstraction reactions, which result in quenching the carbon radicalthrough formation of a new C\H bond, together with formation of anew radical from the hydrogen atom donor. Sulfide and thiol functionalgroups have relatively weak S\H bonds (BDE (RS-H)=92 kcal/mol[84], BDE (RCH2-H)=101 kcal/mol [85]) and a hydrosulfide or thiol

group can rapidly transfer a hydrogen atom to a carbon radical atdiffusion-controlled rates (k≥108 M−1 s−1 [86]). If a free sulfide,alkane thiol, or alkane persulfide was to function as the sulfurdonor for a radical C\S bond forming reaction, then each of thesegroups would be protonated at physiological pH and would prefer-entially quench the carbon radical by hydrogen atom transfer, ratherthan by C\S bond formation. We propose, therefore, that one functionof the [2Fe–2S]2+ cluster is to act as a Lewis acid protecting group thatbinds sulfide but excludes reactive S\H bonds from the active site.

An additional problem with employing a radical mechanism forcatalysis is that radical generation and radical termination both

1221C.J. Fugate, J.T. Jarrett / Biochimica et Biophysica Acta 1824 (2012) 1213–1222

require one-electron events. In an AdoMet radical enzyme such asbiotin synthase, radical generation occurs when one electron istransferred from the [4Fe–4S]+ cluster into the AdoMet sulfonium,resulting in the conversion of a molecule with paired electrons, Ado-Met, into a molecule with a highly reactive unpaired electron, the5′-deoxyadenosyl radical. Likewise, the termination of each radicalsequence must involve a second one-electron event, in which a rad-ical intermediate is quenched by oxidation or reduction to againyield a stable product with completely paired electrons. In general,one electron oxidation or reduction is most easily accomplished bytransition metal ions, which typically have 2 or more easily accessibleredox states. Iron, for example, commonly converts between Fe2+ andFe3+ in biological systems, and iron–sulfur clusters can readily provideor accept one electron to initiate or terminate a radical reaction. Wepropose, therefore, that the [2Fe–2S]2+ cluster also functions as a cata-lytic cofactor that facilitates inner-sphere one-electron oxidation of thesulfide concurrent with formation of the new C\S bond, providing arapid and safe means for terminating the radical reaction sequence atthe end of each half-reaction.

7. Unresolved issues with the biotin synthase reaction mechanism

While there is clear evidence supporting the stepwise conversionof DTB to 9-MDTB and thence to biotin, it is not entirely clear how9-MDTB is bound within the enzyme and prepared for the subsequentring closure reaction. Unfortunately, the only available structure ofbiotin synthase shows DTB in a position that seems unlikely for the9-MDTB intermediate, with C9 near AdoMet and C6 ~0.5 Å furtheraway [66]. Marquet and coworkers found that biotin synthase willconvert exogenous synthetic 9-MDTB to biotin [48], but this reactionis much less efficient. We have observed that this reaction is accom-panied by the production of 10–20 equivalents of 5′-dAH, suggestingthat unproductive quenching of the 5′-deoxyadenosyl radical occursduring most turnovers, possibly by hydrogen atom transfer from the9-MDTB thiol group [78]. Presumably the endogenous [2Fe–2S]2+

cluster present in unreacted biotin synthase is at least partiallyobscuring the correct binding site for 9-MDTB in these studies, andcould be forcing the thiol group toward the 5′-deoxyadenosyl radi-cal. Generation of 9-MDTB during catalytic turnover must result ina significantly different structure that is highly efficient for hydrogenatom abstraction at C6 and subsequent formation of biotin. Identifyingthe structure and electronic state of this catalytic intermediate will fillin an essential piece of mechanistic puzzle.

Those unfamiliar with the biotin synthase story are often shockedat the incredibly slow enzymatic reaction rates observed even inoptimized assays. A single turnover of biotin production occurswith kburst≈0.12 min−1 at 37 °C, and subsequent turnovers proceedat kSS≈0.01 min−1 at 37 °C [79]. The kinetics of formation and decayof 9-MDTB suggest that intermolecular formation of the C9\S bondis at least 5-fold slower than intramolecular formation of the C6\Sbond. Among the events required for formation of the C9\S bond,abstraction of a hydrogen atom from the C9 methyl group standsout as a particularly difficult chemical transformation. If this hydro-gen atom transfer step is the rate-limiting step in catalysis, onemight expect a considerable primary kinetic isotope effect (KIE). Inpreliminary studies with mixtures of unlabeled DTB and (2H3-9-methyl)-DTB, we observe almost no reactivity with the 2H labeledsubstrate, and can roughly estimate the KIE≥40 (Fugate and Jarrett,unpublished results). It has been suggested that biotin synthase maybe a slow enzyme because it has not been kinetically optimized, dueto a lack of selective evolutionary pressure to produce large amountsof biotin. However, a very large KIE on hydrogen atom transfer indi-cates that the C9 dethiobiotinyl carbon radical is a very high energyintermediate, and that the transition state for hydrogen atom trans-fer is of even higher energy. Optimized or not, perhaps the slow

turnover simply reflects Nature's best attempt at catalyzing a nearlyimpossible biochemical reaction.

Acknowledgements

This work was supported by the National Science Foundation(MCB 09-23829 to J.T.J.).

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