Evidence from Mössbauer Spectroscopy for Distinct [2Fe-2S]2+ and [4Fe-4S]2+ Cluster Binding Sites in Biotin Synthase from Escherichia coli

Biotin synthase (BS) catalyzes the substitution of a sulfur atom for hydrogen at two saturated carbon atoms in the substrate dethiobiotin; this is an unprecedented reaction in biochemistry.1 In vitro biotin synthesis is catalyzed by BS in the presence of S-adenosylmethionine (AdoMet) and reduced flavodoxin2 and generates methionine and 5′-deoxyadenosine,3 indicating that biotin synthase is an AdoMet-dependent radical enzyme. In general, AdoMet-dependent radical enzymes4 are active containing a [4Fe-4S]2+/+ cluster that is intimately involved in AdoMet cleavage and radical generation.5 This cluster is bound within a conserved CxxxCxxC sequence motif common to all AdoMet-dependent radical enzymes.6 However, BS from E. coli is purified containing only a stable [2Fe-2S]2+ cluster,7 and isotope labeling suggests that the role of this cluster is to provide sulfur for the biotin thioether ring.8 Thus two potentially conflicting roles for FeS clusters have been proposed in biotin synthase. The FeS clusters in BS are remarkably pliable. While the aerobically purified enzyme is a dimer that contains two [2Fe-2S]2+ clusters,7,9 the protein can be chemically converted to contain primarily [4Fe-4S]2+ or [4Fe-4S]+ clusters by strong chemical reduction with dithionite in the presence or absence, respectively, of 60% ethylene glycol.7,9–11 Reconstitution under milder conditions yields protein that appears to contain ~1:1 [2Fe-2S]2+:[4Fe-4S]2+ clusters, as judged by UV/visible spectra and chemical analysis.12 Comparison of the catalytic activity of various cluster reconstitution states of BS suggests that this latter state, with two different clusters present, is the most active for a single turnover in the absence of additional iron and sulfide.13 However, the precise cluster state of this active enzyme preparation remains ambiguous. Since BS is a homodimer,14 this enzyme could contain one [2Fe-2S]2+ and one [4Fe-4S]2+ cluster per dimer, both bound within the conserved cluster binding site in opposing monomers. Alternatively, this enzyme could contain one [2Fe-2S]2+ and one [4Fe-4S]2+ cluster per monomer, for a total of 4 clusters per dimer bound in two types of sites. To distinguish these potential cluster configurations, we turned to Mossbauer spectroscopy using differentially substituted 57Fe protein. When BS is expressed in E. coli grown in minimal media supplemented with 57FeCl3,15 the purified brown-red protein (57Fe-BS, B) contains 1.9 Fe/monomer17 and exhibits a UV/visible absorption band at 452 nm consistent with the presence of [2Fe-2S]2+ clusters.7 The Mossbauer spectrum16 (Figure 1) indicates the protein contains ≥98% [2Fe-2S]2+ clusters (Table 1), with no evidence of [4Fe-4S]2+ clusters. The isomer shift (0.27 mm/s) and quadropole splitting (0.47 mm/s) are similar to values previously reported for BS apoprotein chemically reconstituted with [2Fe-2S]2+ clusters. When this protein is reduced with dithionite in a buffer containing 60% ethylene glycol,7 the reduced [2Fe-2S] clusters dissociate from the protein and reassemble as [4Fe-4S]2+ clusters.11 The Mossbauer spectrum of undesalted protein prepared according to this method17 (D) contains 92% of cluster-associated Fe in a single species with parameters (δ= 0.42 mm/s, ΔEQ = 1.00 mm/s) that are consistent with a [4Fe-4S]2+ cluster. A small amount of [2Fe-2S]2+ cluster remains (~8% of total cluster Fe) in addition to Fe2+ in both S and O/N coordination environments, presumably present as a buffer contaminant due to only partial cluster reassembly. Similar results have been observed by Trautwein and coworkers examining the conversion of [2Fe-2S]2+ to [4Fe-4S]2+ clusters following dithionite reduction of reconstituted BS apoprotein.9 Figure 1 Mossbauer spectra of biotin synthase: (A) (57Fe)-BS reconstituted with 57FeCl3, Na2S, and DTT,12 (B) initial (57Fe)-BS containing only [2Fe-2S]2+ clusters, (C) (57Fe)-BS reconstituted with unlabeled FeCl3, Na2S, and DTT, (D) (57Fe)-BS containing ... Table 1 Sample Composition and Mossbauer Parameters Although it was originally postulated that this [4Fe-4S]2+ cluster occupied the same site as the original [2Fe-2S]2+ cluster, under milder reducing conditions the protein can be reconstituted with a [4Fe-4S]2+ cluster without disturbing the original [2Fe-2S]2+ cluster.12 When 57Fe-BS is reconstituted with 57FeCl3 and Na2S using dithiothreitol (DTT) as reductant, the Mossbauer spectrum (A) shows the presence of both cluster types with a ratio of ~1:0.8 [2Fe-2S]2+:[4Fe-4S]2+ clusters. This sample also contains significant quantities of mononuclear Fe, as judged by both the Mossbauer spectrum and Fe analyses;17 we have had difficulty consistently removing excess Fe by gel filtration chromatography following chemical reconstitution. Similar mixed cluster spectra were previously obtained for 57Fe-reconstituted BS following reduction and partial air oxidation, although the authors attribute the varying mixtures of clusters to conversion of [4Fe-4S]2+ to [2Fe-2S]2+ clusters upon exposure to air.9 Chemical analysis of the Fe and S2− content of protein containing both [2Fe-2S]2+ and [4Fe-4S]2+ clusters suggested that there was one of each cluster type per monomer.12 This would only be possible if there are two distinct cluster binding sites in each BS monomer. Since mild reduction leaves the original [2Fe-2S]2+ cluster untouched, we used differential substitution with 57Fe to test for separation of the [2Fe-2S]2+ and [4Fe-4S]2+ cluster sites. When 57Fe-BS was reconstituted with unlabeled FeCl3, Na2S, and DTT, the chemical analysis17 and the UV/visible spectrum of the resulting protein (not shown) indicated the presence of both [2Fe-2S]2+ and [4Fe-4S]2+ clusters, while the Mossbauer spectrum (C) showed that only the [2Fe-2S]2+ cluster contains 57Fe. If the [4Fe-4S]2+ cluster were built up by addition of unlabeled Fe to the 57Fe-labeled [2Fe-2S]2+ cluster site, then the Mossbauer spectrum should show half of the 57Fe present in a [4Fe-4S]2+ environment. Since this was not observed, the [4Fe-4S]2+ cluster must be assembled at a separate site from the original [2Fe-2S]2+ cluster. In a complementary experiment, unlabeled BS was reconstituted with 57FeCl3, Na2S, and DTT. Again, the UV/visible spectrum showed both cluster types, but the Mossbauer spectrum (E) shows >90% of the total cluster-associated 57Fe was present in [4Fe-4S]2+ clusters. This demonstrates the stability of the original [2Fe-2S]2+ cluster to exchange with free 57Fe during the reconstitution process and again indicates that the [4Fe-4S]2+ cluster is bound at a unique site. BS contains six conserved cysteine residues. Resonance Raman spectra of BS purified with [2Fe-2S]2+ clusters show a strong B3ut vibrational mode at 301 cm−1 that indicates a 3 S and 1 O/N coordination environment,7 while spectra of reduced BS containing only [4Fe-4S]2+ clusters are more amibiguous, with an A1b breathing mode at 338 cm−1 that lies between typical values for a 4 S coordination environment (~335 cm−1) vs. a 3 S and 1 O/N coordination environment (~340–342 cm−1).7,18 Mutagenesis data implicates the conserved CxxxCxxC motif consisting of Cys53, Cys57, and Cys60 as the binding site for the [4Fe-4S]2+ cluster.19,20 Mutation of any of these residues to alanine prevents reconstitution of a [4Fe-4S]2+ cluster and abolishes activity,19 but leaves the initial [2Fe-2S]2+ cluster intact.20 In contrast, mutation of the other conserved cysteine residues, Cys97, Cys128, and Cys188, to alanine results in proteins that can be reconstituted with a [4Fe-4S]2+ cluster but are still inactive for biotin synthesis.19 We propose that these three cysteine residues are ligands to the [2Fe-2S]2+ cluster. The ability of BS to simultaneously accommodate both [4Fe-4S]2+ and [2Fe-2S]2+ clusters is consistent with unique roles for these clusters in facilitating AdoMet reductive cleavage and biotin sulfur insertion.13