Assembly and Functional Role of the Iron-Sulfur Clusters in Respiratory Complex I

• Applicant: Professor Dr. Thorsten Friedrich
• Subject Area: Biochemistry
• Term: from 2016 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 311771253

Respiratory complex I plays a central role in cellular energy metabolism by coupling NADH oxidation to proton translocation. It is a major source of reactive oxygen species, implicating a role in ageing and its dysfunction was suggested to be related to human neurodegenerative diseases. The complex consists of a peripheral arm catalyzing electron transfer and a membrane arm pumping protons across the membrane. The coupling of the two processes is unknown. Electrons enter the complex at the tip of the peripheral arm and are transported to the quinone binding site at the interface of the two arms by a chain of seven iron-sulfur (Fe/S) clusters. Proteins involved in the assembly of the clusters into the complex are not yet known. Another Fe/S cluster is not part of the electron transfer chain, but it is located in electron transfer distance to the primary electron acceptor. It is strictly conserved but its function is not understood. The most distal cluster of the chain is coordinated by a unique binding motif comprising two adjacent cysteine residues, which might enable the cluster to operate as energy converter. A protein variant lacking this cluster is associated with the inducible lysine decarboxylase (CadA), which in turn interacts with two other proteins of a protein family involved in the assembly and repair of metal clusters.In the second period of the priority program, we will identify other proteins involved in the incorporation of Fe/S clusters into complex I with an emphasis on the incorporation of [4Fe4S] clusters. The role of additional factors will be established and their importance for the maturation of lipoyl synthase will be investigated. The function of the ‘off-pathway’ Fe/S cluster for the complex will be determined by using mutants in which the cluster cannot be reduced. We propose that this cluster regulates the electron input into the complex in response to the redox state of the quinone pool of the membrane. We have generated a variant in which the most distal cluster of the chain is coordinated by a regular binding motif. If the unique ligation of the cluster is a prerequisite for energy conversion, the variant should not be able to pump protons across the membrane. This cluster might need special help to be included into the complex due to its unique binding motif. Because a variant lacking this cluster is associated with CadA, we speculate that CadA is involved in the repair or insertion of this cluster. We will determine the binding of CadA to complex I by electron cryo-microscopy. The contribution of two further proteins interacting with CadA to the assembly of the complex will be investigated by characterizing the complex in the corresponding deletion mutants. We will produce the complex from the chromosomal genes and from an overproduction plasmid to clarify the role of these proteins. We propose that these proteins enable the accessibility to the cluster binding site for yet unknown Fe/S cluster carrier proteins.

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