Protein expression and characterization for systems involved in the biogenesis of iron sulfur proteins

Iron-sulfur clusters are essential cofactors found in all kingdoms of life; they had unique functional roles throughout evolution. These clusters, which are the second major form of complex iron cofactors in biology, are ubiquitous in all organisms, playing a key role in several biological pathways. Mutations on the protein involved in the iron-sulfur clusters biosynthesis pathway are associated with a group of multiple mitochondrial dysfunction syndromes (MMDS). These severe diseases could give infantile encephalopathy, lactic acidosis, leukodystrophy and death in early childhood. In human cells, cluster biosynthesis involves three different types of machinery: ISC (iron-sulfur cluster assembly machinery) located in the mitochondria, CIA (cytosolic iron-sulfur cluster assembly machinery) located in the cytosol and ISC export machinery located in the mitochondria inner membrane. The aim of the thesis was the deep investigation of the third step of the ISC assembly machinery. Indeed, using NMR, UV-vis and CD-vis spectroscopies in combination with size exclusion chromatography and multi-angle light scattering we assessed the key role of NFU1, ISCA1, ISCA2, FDX2 and LIAS in the above-mentioned machinery. Moreover, clinical BOLA3 Cys59Tyr mutation involved in the MMDS diseases has investigated at the atomistic and molecular levels. The gained data elucidated fundamental molecular details in the [4Fe-4S] cluster maturation and transfer to apo recipient proteins along the third step of ISC assembly machinery.