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.
