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

近年来，钼辅因子(MoCO)的生物合成与铁硫团簇(Fe-S)的组装有直接的联系。在大肠杆菌中，MOCO的生物合成因此直接依赖于铁-S簇或铁-S簇组装机械在几个水平上的存在。在MOCO生物合成的第一步，MOAA蛋白需要两个[4Fe-4S]簇才能激活。此外，大肠杆菌中的大多数钼酶都含有大量的Fe-S簇，这些簇参与了分子内的电子转移反应，对酶的活性是必不可少的。此外，还发现L-半胱氨酸脱硫酶ISCS是Fe-S簇组装的主要参与者，它可以动员硫合成MoCO中存在的二硫烯基。此外，参与MOCO生物合成的大多数钼酶和蛋白质的表达受到富马酸和硝酸盐还原的转录调节因子FNR的调节。在厌氧条件下，铁-S簇合物本身的活性直接依赖于铁-S簇合物的有效性，因此，当铁-S簇合物没有组装时，不合成MoCO，也不表达钼酶。需要解决的主要问题是铁的可利用性和Fe-S簇团组装如何调节MoCO的生物合成和钼酶的活性。除了基因调控水平外，还将在蛋白质水平上进行研究。我们将通过FNR来揭开钼酶复杂的调控网络。具体地说，我们计划破译细胞内铁的转运和铁-S簇向FNR和选定的钼酶的移交。特别的目的是确定特定地将[2Fe-2S]簇和[4Fe-4S]簇插入钼酶中的蛋白质。根据硫和铁/铁-S簇的可用性，将确定在选定的钼酶翻译效率水平上调节基因表达的新因素。其中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依赖于MOCO和铁的torCAD基因的表达。基因调控水平的研究以及对蛋白质组和金属组学研究的补充，包括高分辨率透明天然电泳、2D Blue天然NLC-MS/MS、全细胞穆斯堡尔光谱和EPR光谱等方法，以及利用微型热电泳法和荧光共振能量转移(FRET)研究蛋白质-蛋白质相互作用的方法，将在SPP的协作框架内应用。总之，我们的分析将有助于揭示复杂的Fe-S簇网络，该网络对于在大肠杆菌中生产活性钼酶是必不可少的。

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.