Escherichia coli biotin synthase produces selenobiotin. Further evidence of the involvement of the [2Fe-2S]2+ cluster in the sulfur insertion step.

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(NH4)2(SO4)2 and Na2S 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 FeCl3 and Na2Se to yield a [2Fe-2Se]2+ cluster. This enzyme was assayed in vitro with Na2Se in place of Na2S 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 Na2S. The [2Fe-2Se]2+ enzyme was additionally assayed with Na2S,...