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

近年来，钼辅因子（Moco）的生物合成与铁硫（Fe-S）簇的组装之间存在着直接的联系。因此，在大肠杆菌中，Moco的生物合成直接依赖于Fe-S簇或Fe-S簇组装机制在几个层面上的组件的存在。在Moco生物合成的第一步，MoaA蛋白需要两个[4Fe-4S]簇才能发挥活性。此外，大肠杆菌中的大多数钼酶含有大量Fe-S簇，这些簇参与分子内电子转移反应，对酶的活性至关重要。此外，l -半胱氨酸脱硫酶IscS作为Fe-S簇组装的主要参与者也被确定为调动硫来合成Moco中存在的二噻吩基团。此外，参与Moco生物合成的大多数钼酶和蛋白质的表达受富马酸盐和硝酸盐还原转录调控因子FNR的调控。在厌氧条件下，FNR本身的活性直接依赖于Fe-S簇的可用性，因此，当Fe-S簇没有组装时，Moco不会合成，钼酶也不会表达。需要解决的主要问题是铁的有效性和Fe-S簇组装如何调节Moco生物合成和钼酶活性。除了基因调控水平外，研究还将在蛋白质水平上进行。我们将通过FNR揭示钼酸酶的复杂调控网络。具体来说，我们计划破译细胞内铁运输和Fe-S簇移交给FNR和选定的钼酶。特别的目的是鉴定特异性地将[2Fe-2S]簇和[4Fe-4S]簇插入钼酶的蛋白质。研究人员将发现一些新的调控基因表达的因子，这些因子依赖于硫和铁/铁- s簇的可用性，从而影响所选钼酸酶的翻译效率。一个特别的重点是研究大肠杆菌中编码TMAO还原酶的操纵子的调控，因为我们发现了一种新的Moco和铁依赖性表达的torCAD基因。基因调控水平的研究和蛋白质组学和金属组学的补充研究，包括高分辨率原生电泳、2D蓝色原生nLC-MS/MS、全细胞Mössbauer光谱和EPR光谱等方法，以及利用微尺度热电泳和荧光共振能量转移（FRET）的蛋白质-蛋白质相互作用研究，将在SPP的合作框架内应用。我们的分析将揭示复杂的Fe-S簇网络，这对大肠杆菌中活性钼酶的产生至关重要。

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.