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

厌氧细菌和古细菌利用同源酶将二氧化碳和H2转化为乙酰辅酶a和甲烷，这些酶决定了地球上自生命诞生以来的全球碳循环。近年来，我们发现并研究了中心酶乙酰辅酶a合成酶（ACS）的Ni插入酶。一种或多种成熟酶是已知的所有其他中心镍酶；到目前为止，还没有发现支持Ni成熟的酶，即Ni-[4Fe4S]簇，称为簇A。我们可以证明，成熟蛋白CooC2（一种mind家族atp酶）与缺镍的ACS形成特异性复合物，该蛋白复合物以高亲和力结合两个Ni离子，最后通过在复合物中添加Mg-ATP来释放活性ACS。生物信息学分析表明，CooC ATPases可分为三个亚类：一个亚类包含一氧化碳脱氢酶[NiFe4S4]中心成熟的ATPases；一个子类，以CooC2为第一个功能描述的例子，由负责ACS成熟的atp酶组成；第三个子类的作用是未知的。在第一个资助期，我们希望研究：(1)来自古细菌的同源酶是否与细菌的CooC2蛋白具有相同的功能。这是特别有趣的，因为来自古细菌的ACS在大小和与细菌酶的CODH和CoFeSP的关联上不同：与细菌酶相比，它缺少n端结构域，但存在于2.5 MDa的多酶复合物中。我们想研究（II）， CooC2-apoACS复合体对Ni的高亲和力是如何产生的，ATP在成熟过程中发挥了什么作用。除了apo-和holo-ACS，我们还可以生产两种稳定的成熟中间体，这给了我们难得的机会来跟踪复杂的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是胞质铁/S簇成熟机制的关键复合物。为了实现这三个目标，SPP的整合和与SPP其他小组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.