Biotin Synthase: A role for the FeS cluster assembly chaperone HscA in regenerating the [2Fe‐2S] cluster substrate

Abstract

Biotin synthase is an is an S‐adenosylmethionine (AdoMet) radical enzyme that catalyzes the addition of sulfur to dethiobiotin, generating the thiophane ring of biotin. Biotin synthase contains two FeS clusters: a [4Fe‐4S]2+ cluster is involved in radical generation via reductive cleavage of the AdoMet sulfonium, and a [2Fe‐2S]2+ cluster provides the sulfur for the biotin thiophane ring. During a single turnover in vitro, the [2Fe‐2S]2+ cluster is destroyed and the enzyme rendered inactive, although in vivo data suggest that each enzyme molecule undergoes multiple turnovers. Using equilibrium binding methods, we report that the FeS cluster chaperone HscA forms a tight complex with biotin synthase. Further, a 1:1 complex of biotin synthase and HscA also binds the FeS cluster scaffold protein IscU, generating a 3‐way complex. Using mass spectrometry methods, we have identified 3 loop regions near the biotin synthase active site that bind to 3 separate sites on HscA. We suggest a specific role for HscA in targeting IscU to low‐abundance iron‐sulfur proteins such as biotin synthase, facilitating regeneration of the active FeS clusters.