Crosstalk of iron-sulfur cluster assembly, metal homeostasis and the biosynthesis of molybdoenzymes

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In recent years it has become evident that the biosynthesis of the molybdenum cofactor (Moco) and the assembly of iron-sulfur (Fe-S) clusters are directly connected to each other. In Escherichia coli, Moco biosynthesis thereby directly depends on the presence of Fe-S clusters or components of the Fe-S cluster assembly machinery at several levels. In the first step of Moco biosynthesis, the MoaA protein requires two [4Fe-4S] clusters for activity. Further, most molybdoenzymes in E. coli harbor numerous Fe-S clusters that are involved in intramolecular electron transfer reactions and are essential for the activity of the enzymes. In addition, the L-cysteine desulfurase IscS as major player for Fe-S cluster assembly has also been identified to mobilize the sulfur for the synthesis of the dithiolene group present in Moco. In addition, the expression of most molybdoenzymes and proteins involved in Moco biosynthesis is regulated by the transcriptional regulator for fumarate and nitrate reduction, FNR. The activity of FNR itself is directly dependent on the availability of Fe-S clusters under anaerobic conditions, consequently, Moco is not synthesized and molybdoenzymes are not expressed when Fe-S clusters are not assembled.The main questions to be solved are how iron availability and Fe-S cluster assembly regulates Moco biosynthesis and molybdoenzyme activity. The studies will be performed on the protein level in addition to the gene regulation level. We will unravel the complex regulatory network of molybdoenzymes by FNR. Specifically, we plan to decipher the intracellular iron trafficking and Fe-S cluster handover to FNR and selected molybdoenzymes. Particular aims are to identify the proteins that specifically insert [2Fe-2S] clusters and [4Fe-4S] clusters into molybdoenzymes. Novel factors that regulate gene expression on the level of translation efficiency of selected molybdoenzymes in dependence on the sulfur- and iron/Fe-S cluster availability will be identified. One particular focus is to study the regulation of the operon encoding TMAO reductase in E. coli, since we identified a novel Moco and iron-dependent expression of the torCAD genes.Studies on the level of gene regulation and complementing proteomic and metallomic studies including methods like high resolution clear native electrophoresis, 2D blue native nLC-MS/MS, whole cell Mössbauer spectroscopy and EPR spectroscopy, in addition to protein-protein interaction studies using microscale thermophoresis and fluorescence resonance energy transfer (FRET) will be applied within the collaborative framework of the SPP. In total, our analyses will shed light into the complex Fe-S cluster network that is essential for the production of active molybdoenzymes in E. coli.