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 Mössbauer 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 Mössbauer 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 Mössbauer 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, ΔE_Q = 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.

Mössbauer 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 primarily [4Fe-4S]²⁺ clusters, generated by reduction with Na₂S₂O₄⁷ in the presence of ⁵⁷FeCl₃ and Na₂S in 60% ethylene glycol, (E) unlabeled BS reconstituted with ⁵⁷FeCl₃, Na₂S, and DTT. Model spectra used to fit spectrum A are shown at the top.

Table 1.

Sample Composition and Mössbauer Parameters

	57Fe present in each environment (% total)a
Sample	[2Fe-2S]2+	[4Fe-4S]2+	Fe2+S4	Fe2+(O/N)5/6
A	24	40 (0.83)b	17	19
B	98	0	0	2
C	100	0	0	0
D	5	70	3	22
E	8	85	0	7
δ(mm/s)	0.27	0.42	0.63	1.08
ΔEQ (mm/s)	0.49	1.00	2.80	2.60

^aCalculated from spectra in Figure 1.

^b[4Fe-4S]²⁺/[2Fe-2S]²⁺ ratio.

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 Mössbauer 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 Mössbauer 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 Mössbauer 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 Mössbauer 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 B_3u^t 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 A₁^b 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

Biotin synthase is an AdoMet-dependent radical enzyme that catalyzes the insertion of an FeS cluster-derived sulfur atom into dethiobiotin. The dimeric enzyme is purified containing one [2Fe-2S]²⁺ cluster per monomer, but is most active when reconstituted with an additional [4Fe-4S]²⁺ cluster per monomer. Using Mössbauer spectroscopy coupled with differential reconstitution of each cluster with ⁵⁷Fe, we show that the reconstituted enzyme has ~1:1 [2Fe-2S]²⁺ and [4Fe-4S]²⁺ clusters, and that the [4Fe-4S]²⁺ cluster is assembled at an alternate site not previously occupied by the [2Fe-2S]²⁺ cluster. These data suggest that biotin synthase is evolved to simultaneously accommodate two different clusters with unique roles in catalysis.