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Iron-Sulfur-Clusters are some of the most important and versatile cofactors in nature. Many essential metabolic pathways, like the respiratory chain or the citrate cycle are directly dependent on iron-sulfur-cluster containing enzymes. However the toxicity of free iron and sulfide required the development of biosynthetic pathways that minimize the concentrations of free iron or sulfide. This led to the development of evolutionary highly conserved biosynthetic machineries for the construction of iron sulfur-clusters that build upon three essential core components: A scaffold-protein on which the cluster is assembled, a cysteine desulfurase, which catalyzes sulfur mobilization and an iron donating protein, most likely a frataxin homolog.
In this study the core components of the Bacillus subtilis iron sulfur-cluster biosynthesis system weres characterized. After the identification of the putative biosynthesis system via BLAST analysis, the function of the SufU scaffold protein was identified using deletion mutants. SufU was then further characterized by in vitro studies. We could show that reduced expression of SufU led to a severe growth phenotype of Bacillus subtilis and a reduced activity of iron sulfur proteins. Further it could be shown that recombinant SufU was able to bind an Fe/S-cluster and transfer this cluster to apo-Fe/S-proteins.
In addition to the scaffold protein the second core component of the biosynthesis system was characterized: the cysteindesulfurase SufS. It could be shown that purified SufS is inactive and needs to be activated by apo-SufU. The activity is dependent on both the cysteine and SufU concentration and the reaction follows a ping-pong-reaction mechanism as shown by kinetic studies. Furthermore it was found that cysteine 41 of SufU plays an important role in the sulfur transfer mechanism between SufS and SufU.
In the last part of this work the putative iron-binding protein Fra (YdhG) was characterized, which is a structural homolog of human frataxin. The deletion of the gene encoding Fra in B. subtilis led to a severe growth Phenotype and reduced activity of iron sulfur proteins, indicating a greater disturbance of the iron homeostasis. In vitro studies showed that Fra was able to bind iron and acts as an iron donating protein during iron sulfur cluster assembly on SufU.