Escherichia coli Biotin Synthase Produces Selenobiotin. Further Evidence of the Involvement of the [2Fe-2S]²⁺ Cluster in the Sulfur Insertion Step

Abstract

Biotin synthase, a member of the "radical SAM" family, catalyzes the final step of the biotin biosynthetic pathway, namely, the insertion of a sulfur atom into dethiobiotin. The as-isolated enzyme contains a [2Fe-2S](2+) cluster, but the active enzyme requires an additional [4Fe-4S](2+) cluster, which is formed in the presence of Fe(NH(4))(2)(SO(4))(2) and Na(2)S in the in vitro assay. The role of the [4Fe-4S](2+) cluster is to mediate the electron transfer to SAM, while the [2Fe-2S](2+) cluster is involved in the sulfur insertion step. To investigate the selenium version of the reaction, we have depleted the enzyme of its iron and sulfur and reconstituted the resulting apoprotein with FeCl(3) and Na(2)Se to yield a [2Fe-2Se](2+) cluster. This enzyme was assayed in vitro with Na(2)Se in place of Na(2)S to enable the formation of a [4Fe-4Se](2+) cluster. Selenobiotin was produced, but the activity was lower than that of the as-isolated [2Fe-2S](2+) enzyme in the presence of Na(2)S. The [2Fe-2Se](2+) enzyme was additionally assayed with Na(2)S, to reconstitute a [4Fe-4S](2+) cluster, in case the latter was more efficient than a [4Fe-4Se](2+) cluster for the electron transfer. Indeed, the activity was improved, but in that case, a mixture of biotin and selenobiotin was produced. This was unexpected if one considers the [2Fe-2S](2+) center as the sulfur source (either as the ultimate donor or via another intermediate), unless some exchange of the chalcogenide has taken place in the cluster. This latter point was seen in the resonance Raman spectrum of the reacted enzyme which clearly indicated the presence of both the [2Fe-2Se](2+) and [2Fe-2S](2+) clusters. No exchange was observed in the absence of reaction. These observations bring supplementary proof that the [2Fe-2S](2+) cluster is implicated in the sulfur insertion step.

The Iron-Sulfur Cluster Proteins Isa1 and Isa2 Are Required for the Function but Not for the De Novo Synthesis of the Fe/S Clusters of Biotin Synthase in Saccharomyces cerevisiae

“…There is a general agreement that the 4Fe/4S cluster of the active form of biotin synthase mediates the electron transfer to SAM, while the 2Fe/2S cluster is considered to be the sulfur donor (11,22,24,34,41,59). On the basis of experiments in vitro, biotin synthase has been proposed to function as a reactant that is capable of performing only a single sulfur insertion per reaction cycle (11).…”

Structures of lipoyl synthase reveal a compact active site for controlling sequential sulfur insertion reactions

“…These distances allow formation of an octanoyl C6 radical and makes quenching this carbon centered radical by reaction with auxiliary cluster sulfide chemically feasible. Another member of the RS superfamily, biotin synthase, catalyzes a mechanistically related sulfur insertion reaction using an [2Fe-2S] cluster as the sulfur donor [21, [55][56][57][58]. In the biotin synthase crystal structure, which includes the proposed sulfur donor [2Fe-2S] cluster and the substrate dethiobiotin [19], the relevant distances (5'-C of SAM to DTB C9 and DTB C9 to the proximal sulfur of the [2Fe-2S] cluster) are approximately comparable at 3.9 and 4.1 Å respectively.…”

Loss of iron–sulfur clusters from biotin synthase as a result of catalysis promotes unfolding and degradation

“…A [2Fe-2S] 2+ cluster is bound in close proximity to dethiobiotin within the core of the (αβ) 8 barrel fold. Both isotope labeling and spectroscopic data suggest that a sulfide from this cluster becomes incorporated into dethiobiotin [9,13,14], generating the thiophane ring of biotin. However, the cluster is more than just a binding site for sulfide.…”

“…BioB expressed in media containing 35 S-cysteine [11], or apoBioB reconstituted with Fe 3+ and 34 S-sulfide or selenide [12][13][14], results in an enzyme with an isotope-or selenide-labeled [2Fe-2S] 2+ cluster whose turnover produces biotin that incorporates the heavy-atom label in 60-80% yield. When turnover of BioB is monitored by UV/visible spectroscopy, the absorption band at ∼450 nm associated with the [2Fe-2S] 2+ cluster disappears at a rate that is similar to the rate of biotin formation [9].…”