CooC2/AcsF and Cfd1/Nbp35: maturation of complex Fe/S-clusters by MinD-type ATPases

厌氧细菌和古细菌使用同源酶将CO2和H2转化为乙酰辅酶A和甲烷，这决定了自生命诞生以来地球上的全球碳循环。近年来，我们发现并研究了一个镍插入酶的中心酶乙酰辅酶A合成酶（ACS）。虽然已知一种或多种成熟酶用于所有其他中心Ni酶;但迄今为止还没有描述支持Ni成熟的酶，Ni-[4Fe4S]簇，称为簇A。我们可以表明，成熟蛋白CooC2，一个MinD家族的ATP酶，形成一个特定的复合物与镍缺乏ACS，该蛋白质复合物结合两个镍离子具有高亲和力，最后，活性ACS是通过添加Mg-ATP的复合物释放。生物信息学分析表明，CooC ATP酶可以分为三个亚类：一个亚类包含用于一氧化碳脱氢酶[NiFe4S4]中心成熟的ATP酶;一个亚类，以CooC2作为功能描述的第一个例子，由负责ACS成熟的ATP酶组成;第三个亚类的作用未知。在第一个资助期内，我们想研究：（I）来自古细菌的同源酶是否具有与细菌CooC2蛋白相同的功能。这是特别令人感兴趣的，因为来自Escherichia的ACS在大小和与来自细菌酶的CODH和CoFeSP的关联方面不同：与细菌酶相比，它缺少N-末端结构域，但存在于2.5 MDa的多酶复合物中。我们想研究（II），如何在CooC2-apoACS复合物中产生对Ni的高亲和力，以及ATP在成熟中发挥何种作用。除了apo和holo-ACS，我们可以产生两个稳定的成熟中间体，让我们有机会跟随复杂的Ni，Fe，S-簇的逐步组装。此外，（III）我们想比较CooC2与不同类型的MinD金属插入酶Cfd1/Nbp35的插入策略，Cfd1/Nbp35是胞质Fe/S簇成熟机制的关键复合物。为了实现这三个目标，融入最高人民法院以及与最高人民法院其他团体的互动是必不可少的。

Anaerobic bacteria and archaea use homologous enzymes to convert CO2 and H2 to acetyl-CoA and methane, which determine since the dawn of life the global carbon cycle on earth. In recent years, we have discovered and investigated a Ni insertase for the central enzyme acetyl-CoA synthase (ACS). While one or more maturation enzymes are known for all other central Ni enzymes; so far no enzyme supporting the maturation of Ni,Ni-[4Fe4S] cluster, called Cluster A, has been described. We could show that the maturation protein CooC2, a MinD-family ATPase, forms a specific complex with the Ni-deficient ACS, that this protein complex binds two Ni ions with high affinity and finally that the active ACS is liberated by the addition of Mg-ATP to the complex. Bioinformatic analyzes suggest that the CooC ATPases can be divided into three subclasses: one subclass contains ATPases for the maturation of the [NiFe4S4] center of the carbon monoxide dehydrogenases; one subclass, with CooC2 as the first example described functionally, consists of ATPases responsible for the maturation of the ACS; the role of the third subclass is unknown. In the first funding period we want to investigate: (I) whether the homologous enzymes from archaea have the same function as the bacterial CooC2 protein. This is particularly interesting since the ACS from Archaea differs in size and association with CODH and CoFeSP from the bacterial enzyme: compared to the bacterial enzymes it misses the N-terminal domain, but is present in a multi-enzyme complex of 2.5 MDa. We want to investigate (II), how the high affinity for Ni is generated in the CooC2-apoACS complex and which role ATP plays in the maturation. In addition to apo- and holo-ACS, we can produce two stable maturation intermediates giving us the rare opportunity to follow the stepwise assembly of a complex Ni, Fe, S-cluster. Furthermore, (III) we want to compare the insertion strategy of CooC2 with a different type MinD metal-insertase, Cfd1/Nbp35, a key complex of cytosolic Fe/S cluster maturation machinery. In order to achieve the three objectives, integration in the SPP and the interaction with other groups of the SPP are indispensable.