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Molecular Understanding of Generation and Trafficking of Mitochondrial [4Fe-4S] Clusters

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

From microbes to man, iron is a ubiquitous component of cellular function. In the biological context, iron comes in several different forms, one of which is an inorganic cofactor where sulfur and iron form small clusters termed iron-sulfur (Fe-S) clusters. The most common clusters, [2Fe-2S] and [4Fe-4S] clusters, are key points in metabolic energy production, and in eukaryotes they are essential for the maintenance of DNA and the production of proteins. An important consideration of these clusters is that they do not spontaneously form inside the cell; they must be strictly synthesized by components of the cell because uncontrolled iron and sulfide concentrations can damage biomolecules leading to cellular dysfunction. Therefore, over the past two decades, much work has been put forth to understand how these clusters are generated in the cell. In vivo studies have generated a model for eukaryotic Fe-S cluster biogenesis with at least 30 proteins, and continued efforts to reconstruct cluster biogenesis in the test tube have also provided molecular insight. This model has been used successfully as a blueprint to pinpoint disrupted Fe-S proteins and pathways that lead to often fatal genetic diseases. Looking to the future, this model could also be highly valuable for the construction or optimization of biotechnologically relevant organisms, which take advantage of the chemical versatility of Fe-S clusters for the production of fine chemicals. A complete model with mechanisms and cluster trafficking pathways, however, remains lacking for the Fe-S biogenesis in the mitochondria (refer to review listed in the next section). The aim of this project, FourFeFourS, was to advance this model by determining the underlying mechanisms of [4Fe-4S] cluster biogenesis in the mitochondrion. Specifically, an in vitro assay to determine the factors and mechanisms for [4Fe-4S] cluster synthesis was pursued. Before this could be done, a key component most likely required for [4Fe-4S] synthesis, namely the source of electrons, needed to be deciphered.

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.
As Fe-S cluster biogenesis is conserved in most eukaryotes, Saccharomyces cerevisiae or Baker’s yeast was chosen as a tractable model organism for knocking out one, two, or all three thiol oxidoreductases, i.e. thioredoxin reductase 1 (T1), thioredoxin 2 (T2), and glutathione reductase (G1). Since the thioredoxin system can partially fulfill the functions of the glutaredoxin system, we focused on mutants with no thiol oxidoreductases in the mitochondrion (deficient in T2G1), none in the cytosol (deficient in T1G1), and a cell lacking all (deficient in T1T2G1) (Figure 1). It was expected that if thiol oxidoreductases are providing electrons for Fe-S biogenesis, that removal of this electron source should disrupt the activities of Fe-S cluster proteins and in turn iron homeostasis.

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).
Our work is the first thorough assessment of the physiological role that thiol oxidoreductases play in cellular Fe-S protein biogenesis (Figure 1). This is an important consideration because the thiol oxidoreductase systems are also responsible for neutralizing harmful reactive oxygen species (ROS), which are known to affect Fe-S clusters and damage proteins and DNA. Determining how the disruption of one system affects the other could be highly valuable in diagnosing disease. The knowledge gained from FourFeFourS advances the model of Fe-S cluster biogenesis, which will inform present and future collaborations in the field of medicine.