Functional interactions of a monothiol glutaredoxin and an iron sulfur cluster carrier protein with the radical SAM enzyme MiaB

The biosynthesis of iron sulfur (FeS) clusters is a highly coordinated process enabled by multi‐protein systems such as Isc and Suf in E. coli . While these systems are believed to encode all factors required for initial cluster assembly and transfer to FeS carrier proteins, accessory factors of undefined function, including the monothiol glutaredoxin, GrxD and the FeS carrier protein NfuA, are located outside of these defined systems. These factors have been suggested to function both as shuttle proteins acting to transfer clusters between scaffold and carrier proteins, and in the final stages of FeS protein assembly by transferring clusters to client FeS apo‐proteins. Here we implicate both of these factors in client protein interactions. We demonstrate specific interactions between GrxD, NfuA and the methylthiolase MiaB, a radical SAM enzyme involved in tRNA maturation. We show that GrxD and NfuA physically interact with MiaB with affinities compatible with an in vivo function. We furthermore demonstrate that NfuA is able to transfer its cluster in vitro to MiaB, while GrxD is unable to do so. The relevance of these interactions was demonstrated by linking the activity of MiaB with GrxD and NfuA in vivo . We observe a severe defect in in vivo MiaB activity in cells lacking both GrxD and NfuA, suggesting that these proteins may play complementary roles in the MiaB catalytic cycle. Supported by NIH award GM088196 to GB.