Role of the [2Fe-2S]2+ Cluster in Biotin Synthase: Mutagenesis of the Atypical Metal Ligand Arginine 260†

Biotin synthase is an iron-sulfur enzyme that catalyzes the formation of the biotin thiophane ring (1-3). The enzyme contains a cuboidal [4Fe-4S]2+ cluster that is coordinated at a unique Fe atom by the methionyl carboxylate and amine of AdoMet1 (4,5). This covalent interaction facilitates the close association of the AdoMet sulfonium with the [4Fe-4S]+ cluster that is thought to be important for the one-electron reductive cleavage of an AdoMet C-S bond, generating methionine and a transient 5′-deoxyadenosyl radical (6). The substrate dethiobiotin lies in van der Waals contact with the AdoMet ribose, and H-atom transfer from C9 of dethiobiotin to the 5′-deoxyadenosyl radical would generate the observed product, 5′-deoxyadenosine, and a substrate-centered carbon radical. Formation of a new C-S bond on the path to biotin presumably involves a reaction of this substrate radical with a sulfur donor; the precise identity of this sulfur donor remains the most significant unanswered mechanistic question. Biotin synthase belongs to the SAM radical superfamily, which includes ca. 20 known AdoMet-dependent radical enzymes and perhaps more than 600 unique unidentified enzymes (7). These enzymes share a FeS cluster binding motif, CxxxCxxC, that is found within an extended loop region and binds the [4Fe-4S]2+/+ cluster (Cys53, Cys57, and Cys60 in BS from E. coli) (5, 8). These enzymes also share somewhat weaker conservation of residues involved in AdoMet binding, including a hydrophobic residue at position 7 of the cluster motif (Tyr59 in BS) that forms a portion of the adenine binding pocket, and Asp/Glu and Asn/Gln residues (Asp155 and Asn153 in BS) that hydrogen bond to the ribose hydroxyl groups (8). In addition to residues shared with other AdoMet radical enzymes, BS sequences share conserved residues involved in binding DTB that includes the motif N(H/N)N(L/I)(D/E), where Asn151 and Asn153 hydrogen bond to the ureido ring of dethiobiotin. Finally, BS contains an essential [2Fe-2S]2+ cluster that is coordinated by Cys97, Cys128, Cys188, and Arg260; this motif was initially not identified because it is spread throughout the protein, with each residue originating from a different β strand within the (αβ)8 barrel that surrounds the active site (5). Mutagenesis studies have suggested that Cys97, Cys128, and Cys188 are critical for binding to the [2Fe-2S]2+ cluster, since replacement of each residue with Ser resulted in the generation of an inactive mutant apoprotein(9). Although vibrational spectroscopy had indicated O/N ligation at the fourth coordination position of this cluster (10, 11), the identification of Arg260 was made only through solution of the crystal structure of the E. coli enzyme (5), and we postulated that this unusual metal ligand must have a unique catalytic or structural role. The closest bridging sulfide of the [2Fe-2S]2+ cluster lies 4.7 A from C9 of dethiobiotin (5). Several experimental results have suggested that a sulfide from this cluster is incorporated as the thiophane sulfur of biotin. When biotin synthase is purified from media supplemented with (35S)-cysteine, the enzyme contains a 35S-labeled [2Fe-2S]2+ cluster and partial turnover results in formation of (35S)-biotin (12). Likewise, when the purified enzyme is stripped of unlabeled FeS clusters and reconstituted with Fe2+ and 34S2-, then the biotin produced incorporates ∼70% of the expected heavy-atom label (13). More recently, partial turnover of BS containing a [2Fe-2Se]2+ cluster, generated by reconstitution with Fe2+ and sodium selenide, produced biotin that contained 45-70% selenium in place of sulfur (14). Transfer of sulfur from the [2Fe-2S]2+ cluster is accompanied by apparent destruction of the cluster, as detected in the optical spectrum by a broad decrease in absorbance centered at ∼450 nm (15), and in the Mossbauer spectrum by a decrease in the narrow quadropole doublet associated with the [2Fe-2S]2+ cluster (δ=0.29 mm/s, ΔEQ=0.51 mm/s) and an appearance of much broader doublets associated with Fe2+ ion (δ=0.73, 0.86 mm/s, ΔEQ=3.36, 3.65 mm/s) (16). The conservation of the atypical guanidinium metal ligand Arg260, together with the implication of the [2Fe-2S]2+ cluster as the probable sulfur donor, suggested that this residue played an important role in the enzyme. We postulated several possible roles: (i) the potential positive charge of the guanidinium could be important in modulating the redox potential of the FeS cluster, (ii) the bidentate nature of a guanidinium functional group could facilitate liberation of sulfide from the cluster during turnover and would therefore be catalytically important, (iii) the long Arg260 side chain could be important for closing β strand 7 around the barrel-shaped active site and would thus be structurally important (5), or (iv) protonation of the guanidine following turnover could facilitate opening the active site to allow cluster assembly and/or repair by the endogenous iron-sulfur cluster assembly system (ISC) of E. coli. To test these hypotheses, we mutated Arg260 to Ala, Cys, His, and Met, and conducted a series of experiments designed to examine changes in the properties of the [2Fe-2S]2+ clusters, the stability of the folded protein with and without the [2Fe-2S]2+ cluster, and the importance of this residue for in vitro and in vivo activity.