Deciphering the influence of the [4Fe–4S] cluster of tRNA thiolation enzymes on tRNA binding

Iron-sulfur clusters [Fe-S] play crucial roles in diverse biological reactions, often serving as prosthetic groups for enzymes. Specifically, certain tRNA-modifying enzymes utilize these clusters to catalyze the thiolation of specific nucleosides. While the participation of [4Fe-4S] clusters in such catalytic processes is known, their potential influence on tRNA binding remains unexplored. In this study, we examine the impact of the cluster on the affinity for tRNA of TtuI from the archeon <i>Methanococcus maripaludis</i>, an enzyme responsible for the formation of 4-thiouridine at position 8 in tRNAs of archaea and bacteria, as well as <i>Escherichia coli</i> TtcA that catalyzes the biosynthesis of 2-thiocytidine at position 32 in bacterial tRNAs. For this purpose, we compare the change of fluorescence properties of judiciously located tryptophans upon tRNA binding between the apo-enzyme (lacking the cluster) and the holo-enzyme (incorporating a reconstituted cluster). Our results indicate that the presence of the [4Fe-4S] cluster does not alter the affinity of the thiolases for tRNA, thus ruling out any direct involvement of the cluster in tRNA binding and emphasizing the purely catalytic role of the [4Fe-4S] cluster in tRNA thiolation.