SmsB-SmsC machinery in <i>Thermococcus kodakarensis</i> functions as an archaeal scaffold mediating Fe-S cluster assembly

The SUF system is one of the multiprotein machineries responsible for iron-sulfur (Fe-S) cluster biogenesis. In bacterial and eukaryotic SUF systems, SufB, SufC, and SufD form a SufBC₂D complex as the scaffold for Fe-S cluster assembly. SMS (SUF-like minimal system), composed only of SufB and SufC homologs (SmsB and SmsC) without a SufD component, has recently been established, and representatives from methanogenic archaea have been experimentally verified to function in Fe-S cluster biogenesis. SMS has been proposed to be an ancestor of the SUF system. The hyperthermophilic archaeon <i>Thermococcus kodakarensis</i> harbors candidate proteins for SmsB and SmsC (<i>Tk</i>-SmsB and <i>Tk</i>-SmsC) encoded by TK0730 and TK0731 genes, respectively. As <i>Tk</i>-SmsB is phylogenetically positioned in a clade distinct from the previously characterized SmsB proteins from methanogens, here, we examined whether <i>Tk</i>-SmsB and <i>Tk</i>-SmsC can also function in Fe-S cluster generation and transfer. <i>Tk</i>-SmsB and <i>Tk</i>-SmsC formed a heterotetrameric SmsB₂C₂ complex. <i>Tk</i>-SmsC displayed ATPase activity, and its catalytic efficiency (<i>k</i>_cat/<i>K</i>_m) increased up to 10-fold upon complex formation with <i>Tk</i>-SmsB. The <i>Tk</i>-SmsB₂C₂ complex could generate Fe-S clusters in the presence of ferric and sulfide ions. The holo-<i>Tk</i>-SmsB₂C₂ complex could transfer the Fe-S clusters to the apo-form of the Fe-S cluster-dependent lipoyl synthase LipS from <i>T. kodakarensis</i>, resulting in a LipS with sulfur insertion activity for lipoyl group biosynthesis. In the presence of cysteine desulfurase and ATP, the apo-<i>Tk</i>-SmsB₂C₂ complex could also generate Fe-S clusters utilizing cysteine as a sulfur donor and activate LipS.IMPORTANCEA representative of an SmsB protein from <i>T. kodakarensis</i> (<i>Tk</i>-SmsB) that lies in a clade phylogenetically distinct from those of previously verified SmsB proteins has been examined. The results demonstrate that <i>Tk</i>-SmsB, along with <i>Tk</i>-SmsC, functions as a scaffold for Fe-S cluster synthesis in <i>T. kodakarensis</i>, adding further support to the proposition that SMS represents the primitive form of the SUF systems widely present in bacteria and eukaryotes.