Periodic Reporting for period 1 - FourFeFourS (Molecular Understanding of Generation and Trafficking of Mitochondrial [4Fe-4S] Clusters)
Reporting period: 2015-06-15 to 2017-06-14
Thiol oxidoreductases are the first enzymes in an electron relay from the electron-rich molecule, nicotinamide adenine dinucleotide phosphate (NADPH), to proteins in need of electrons. Thioredoxins (Trx) and glutaredoxins (Grx) are key intermediate players that facilitate this transfer of electrons (Figure 1). Interestingly, monothiol glutaredoxins Grx3, Grx4, and Grx5 are also known to bind [2Fe-2S] clusters and are involved in Fe-S cluster biogenesis pathways in both the mitochondrion and the cytosol. We reasoned that the monothiol Grxs could be a direct link between thiol oxidoreductases and Fe-S cluster biogenesis. Therefore, efforts were focused on asking the question, if thiol oxidoreductases could be responsible for delivering electron equivalents for [4Fe-4S] cluster synthesis or reduction of disulfide bridges of target Fe/S proteins.
The work carried out on this project has indicated that thiol oxidoreductases most likely do not provide electron equivalents for [4Fe-4S] cluster biogenesis in the mitochondrion. However, by studying all cellular thiol oxidoreductase systems, we could demonstrate a crucial role of redox balance in cytosolic Fe-S protein maturation. These findings have been essential for the advancement of the in vitro reconstitution model.
Using enzyme activity assays and a 55Fe radiolabeling assay to probe [2Fe-2S] and [4Fe-4S] proteins, it was found that mitochondrial Fe-S proteins are more resistant to thiol redox imbalance as compared to the cytosol. For example, the metabolically important [4Fe-4S] cluster protein aconitase was not disrupted in mitochondria while three cytosolic [4Fe-4S] target proteins lost their function. The [2Fe-2S] cluster-binding protein Grx4 maintained its ability to bind iron in both the mitochondrion and the cytosol. These results indicate that the core ISC machinery and [4Fe-4S] cluster synthesis in the mitochondrion is not dependent on thiol oxidoreductases. At least 11 proteins are involved in the cytosolic Fe-S protein assembly (CIA) pathway and it may be possible that one or more of these proteins are redox-sensitive and depends on the thiol oxidoreductase systems for normal function. Current efforts are trying to pinpoint which CIA protein(s) could be responsible for defective cytosolic Fe/S protein biogenesis. Further, we investigate if the [4Fe-4S] target proteins themselves are oxidatively modified in the mutants.
One further piece of evidence for the observation that the core Fe-S biogenesis machinery in the mitochondrion does not require electrons from the thiol oxidoreductase relay is the normal cellular iron levels under our conditions. The expression of genes that regulate levels of iron in the cell are controlled by the transcription factor Aft1/2 that requires Fe-S cluster-bound Grx4 for function. The source of the cluster for Grx4 is the ISC machinery in mitochondria. When cellular iron levels are adequate, Aft1/2 in combination with [2Fe-2S]-Grx4 prevents the expression of genes that import and distribute more iron in the cell. If Fe-S protein biogenesis is disrupted, Grx4 no longer binds a cluster and this is signaled to the cell nucleus to import iron. We did not see this response in any of the thiol oxidoreductase mutants confirming the intactness of the Fe-S cluster biogenesis machinery in the mitochondria.
Findings from this project have been or will be presented at the 2016 Mitochondria and Chloroplasts Gordon Research Conference, the 2017 EMBO workshop on Thiol Oxidation in Toxicity and Signaling, symposia of DFG funded SPP1710 Dynamics of Thiol-based Redox Switches in Cellular Physiology, and symposia of DFG funded SFB987 Microbial Diversity in Environmental Signal Response. The results will be published in the near future in a peer-reviewed molecular biology journal. Funding has also facilitated publication of an invited review on mitochondrial Fe-S biogenesis (Journal of Biological Chemistry, 2017, 292, 12754-12763).