The [4Fe−4S]²⁺ Cluster in Reconstituted Biotin Synthase Binds S-Adenosyl-l-methionine

Abstract

The final step of the biotin biosynthetic pathway is catalyzed by biotin synthase (BioB) and involves the insertion of sulfur into dethiobiotin. The discovery that [2Fe-2S] 2+ clusters in as-isolated BioB can be converted into [4Fe-4S] 2+ clusters under reducing conditions, 1 coupled with the requirement of S-adenosyl-L-methionine (SAM) for activity in vitro and the conserved C-X 3 -C-X 2 -C cluster-binding motif, led to speculation that BioB was a member of the radical SAM family of enzymes, 2 that utilize a [4Fe-4S] 2+,+ cluster to initiate a radical reaction by mediating reductive cleavage of SAM to yield methionine and a 5′-deoxyadenosyl radical. 3 Subsequent spectroscopic, analytical and mutagenesis studies have demonstrated that BioB can accommodate one [4Fe-4S] 2+,+ cluster per monomer, 4 and provided direct evidence that the [4Fe-4S] + cluster is responsible for reductive cleavage of SAM and is ligated by the cysteines in the C-X 3 -C-X 2 -C motif. 5 In this study, the combination of resonance Raman (RR), Mo ¨ssbauer, and EPR have been used to investigate the effect of SAM on the spectroscopic properties of the [4Fe-4S] 2+ cluster in BioB. The results indicate that SAM interacts directly at a unique Fe site of the [4Fe-4S] 2+ cluster in BioB, mimicking the interaction between SAM and the [4Fe-4S] 2+ cluster established in pyruvate-formate lyase activating enzyme (PFL-AE). 6 This suggests a common mechanism of reductive cleavage of SAM for all enzymes in the radical SAM family. 1 The resonance Raman (RR) spectra of [4Fe-4S] 2+ BioB 7 in the absence and presence of SAM are shown in Figure 1. Both spectra are characteristic of [4Fe-4S] 2+ clusters, and the changes in the frequencies of the Fe-S stretching modes induced by SAM are characteristic of binding a nonthiolate ligand at a unique Fe site. This is best illustrated by the symmetric stretching mode of the [4Fe-4S] core which shifts from 338 to 342 cm -1 on addition of SAM, outside the range established for [4Fe-4S] 2+ cluster with complete cysteinyl coordination [333-339 cm -1 ]. 8 For example, analogous spectral changes to those associated with SAM addition to [4Fe-4S] 2+ BioB are observed in Pyrococcus furiosus ferredoxin (Pf Fd) when one cysteine ligand of the all-cysteinyl ligated [4Fe-4S] 2+ cluster in the D14C variant is replaced by an aspartate in the WT protein. 8 The 4.2 K Mo ¨ssbauer spectrum of the [4Fe-4S] 2+ BioB shows a quadrupole doublet (hatched marks, Figure 2a) 9 with parameters (∆E Q ) 1.16 mm/s, δ ) 0.45 mm/s) similar to values reported previously for the [4Fe-4S] 2+ cluster in BioB. 4,5,10 In the presence of SAM, the doublet broadened substantially and changed shape (hatched marks, Figure 2b). A difference spectrum of ab (hatched marks, Figure 2c) reveals in detail the spectral changes caused by the addition of SAM; a quadrupole doublet contained in spectrum a (seen as two troughs in c) has converted into two quadrupole

Radical S-Adenosylmethionine Enzyme Coproporphyrinogen III Oxidase HemN

“…However, for the latter spectrum, a change in the isomer shift of the non-cysteine-ligated iron atom (from ␦ 2 ϭ 0.57 (3) mm/s to ␦ 2 ϭ 0.68 (3) mm/s) was observed, which indicates that AdoMet only binds at one iron of the cluster, namely this unique, non-cysteine-ligated iron atom. AdoMet binding to this unique iron site of the [4Fe-4S] cluster was previously observed by Mössbauer studies of PFL-AE (17) and biotin synthase (18). Furthermore, our results from the Mössbauer spectrum of HemN in the presence of AdoMet are consistent with the observed coordination of AdoMet to the iron-sulfur cluster in the crystal structure of E. coli HemN (5) in which it was observed that AdoMet binds at the iron by means of the carboxyl and the amino group of the methionine part of AdoMet.…”

“…The iron-sulfur clusters of several members of the family have been characterized by electron paramagnetic resonance (EPR), resonance Raman, and Möss-bauer spectroscopy (7)(8)(9)(10)(11)(12)(13)(14)(15)(16). For pyruvate formate-lyase-activating enzyme (PFL-AE) and biotin synthase (BioB), it was shown using Mössbauer spectroscopy that the [4Fe-4S] cluster contains one iron atom, which is not ligated by a cysteine residue, and that AdoMet binds to this special iron site of the cluster (17,18). Later, AdoMet binding was studied in detail by EN-DOR spectroscopy for PFL-AE and lysine 2,3-aminomutase (LAM) (19 -21).…”

Characterization of MOCS1A, an Oxygen-sensitive Iron-Sulfur Protein Involved in Human Molybdenum Cofactor Biosynthesis

“…Thus far two members of the AdoMet-dependent family of FeS-containing radical enzymes have been structurally characterized by x-ray crystallography, biotin synthase (BioB) and coproporphyrinogen III oxidase (HemN) (60,61), and both confirm the mode of binding of AdoMet to the unique iron site of the [4Fe-4S] cluster proposed based on spectroscopic studies (57,58). In the case of biotin synthase, the assembly of two FeS clusters at different binding sites has been demonstrated: an oxygen-sensitive [4Fe-4S] 2ϩ,ϩ cluster ligated by a CX 3 CX 2 C motif that binds AdoMet and an air-stable [2Fe-2S] cluster ligated by three conserved cysteines and an arginine residue in a CX 30 CX 59 CX 71 R arrangement (42,59,(61)(62)(63). While the physiological relevance of the [2Fe-2S] cluster in recombinant biotin synthase is still a subject of debate, it has been shown to be capable of providing the sulfur for biotin formation in a single turnover experiment (64 -66).…”

Anaerobic Sulfatase-maturating Enzymes, First Dual Substrate Radical S-Adenosylmethionine Enzymes

“…Crystallographic studies have demonstrated that AdoMet binds to the unique iron of the active site [4Fe-4S] 2ϩ cluster in radical-AdoMet enzymes via the methionine amide and carboxylate groups (16) and this is manifest by 3-4 cm Ϫ1 upshifts in both dominant bands in the resonance Raman spectrum, i.e. the symmetric breathing mode of the [4Fe-4S] core and the asymmetric stretching mode of the terminal Fe-S(Cys) ligands (17). These bands are observed at 336 and 356 cm Ϫ1 , respectively, in reconstituted anSMEcpe and upshift to 339 and 360 cm Ϫ1 , respectively, on addition of excess AdoMet.…”