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

近年来，已变得明显的是，钼辅因子（Moco）的生物合成和铁硫（Fe-S）簇的组装直接相互连接。因此，在大肠杆菌中，Moco生物合成直接取决于Fe-S簇或Fe-S簇组装机制的组分在几个水平上的存在。在Moco生物合成的第一步中，MoaA蛋白需要两个[4Fe-4S]簇来进行活性。此外，E.大肠杆菌中含有大量的Fe-S簇，它们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已被确定为动员硫用于合成Moco中存在的二硫杂环戊烯基团。此外，参与Moco生物合成的大多数脱氢酶和蛋白质的表达受富马酸盐和硝酸盐还原的转录调节因子FNR的调节。FNR本身的活性直接依赖于Fe-S簇在厌氧条件下的可利用性，因此，当Fe-S簇未组装时，Moco不合成，氧化酶不表达，需要解决的主要问题是铁的可利用性和Fe-S簇组装如何调控Moco的合成和氧化酶活性。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来阐明腺苷酸酶复杂的调控网络。具体来说，我们计划破译细胞内铁运输和Fe-S簇移交给FNR和选定的辅酶。特别的目的是鉴定特异性地将[2Fe-2S]簇和[4Fe-4S]簇插入腺苷酸酶中的蛋白质。新的因素，调节基因表达的水平上的翻译效率的选择依赖于硫和铁/Fe-S簇的可用性？其中一个特别的焦点是研究编码TMAO还原酶的操纵子在E.在基因调控水平的研究和补充蛋白质组学和金属组学研究方面，包括高分辨率透明天然电泳、2D蓝色天然nLC-MS/MS、全细胞穆斯堡尔谱和EPR谱等方法，除了使用微尺度热泳和荧光共振能量转移（FRET）的蛋白质-蛋白质相互作用研究之外，将在SPP的合作框架内实施。总之，我们的分析将揭示复杂的Fe-S簇网络，这是必不可少的生产活性辅酶在大肠杆菌。杆菌

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.