The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron sulfur (FeS) clusters, molybdopterin (MPT), thiamin, lipoic acid, biotin and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners, e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis and ThiI for thiamine biosynthesis/tRNA thiolation, which bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor (Moco) biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes like nitrate reductase were also increased. Contrarily, the activity of all molydoenzymes analyzed was significantly reduced in the ΔtusA mutant. Characterization of the ΔtusA strain under aerobic conditions showed an overall low MPT content and an accumulation of cyclic pyranopterin monophosphate (cPMP). Under anaerobic conditions the activity of nitrate reductase was only 50% reduced, showing that TusA is not essential for Moco biosynthesis. We present a model in which we propose that the direction of sulfurtransfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis, and the overproduction of FeS clusters leads to a modified expression of several genes.