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

• Applicant: Professor Dr. Holger Dobbek
• Subject Area: Biochemistry ; Structural Biology
• Term: from 2016 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 311061912

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.

• DFG Programme: Priority Programmes
• Subproject of: SPP 1927: Iron-Sulfur for Life