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In S. cerevisiae, three distinct protein machineries are responsible for the de novo biogenesis of Fe/S-clusters and their incorporation into target proteins: the mitochondrial ISC- (iron sulfur cluster) machinery, the ISC-export system and the CIA- (cytosolic iron-sulfur-protein assembly) system. The mechanism of the de novo biogenesis of a [2Fe-2S]-cluster by the core components of the ISC-machinery is well characterized on a functional level. On the contrary, little is known about the process of Fe/S-cluster conversion, catalyzed by the late acting ISC-components, or how Fe/S-target proteins are recruited to the ISC-machinery. This thesis focuses on the last step of Fe/S-cluster biogenesis; the incorporation of the Fe/S-cluster into target proteins. The goal of the first project was to analyze the status of the cysteine ligands of apo-Fe/S-proteins prior to the insertion of an Fe/S-cluster. Because the thiol-groups of these cysteines are exposed to the mitochondrial matrix, it seemed likely that the cell would manage to protect them against oxidative damage. Mass spectrometrical analysis of a mitochondrial Fe/S-protein revealed multiple increments in mass of +30 to +32 Da on Fe/S-proteins in cells with defects in the ISC-machinery. These modifications are most likely bridging polysulfides between the cysteine ligands of apo-Fe/S-proteins. Furthermore, these modifications were also found to a lesser degree in wildtype cells grown under standard conditions, and to a higher degree in cells which were cultured in iron poor medium. As the cysteine modifications could also be reductively cleaved, we suggest that the modification of the cysteine ligands of apo-Fe/S-proteins, with a per- or polysulfide, is actually a physiological protection mechanism against oxidation dependent protein degradation.
In the second project, a protein-fragment complementation assay was established in mitochondria of S. cerevisiae in order to characterize interactions between the late acting ISC-components Isa1, Isa2 and Iba57. The data of this Renilla split-luciferase assay indicates a trimeric complex of these three proteins in vivo. The assembly of this complex seems to be dependent of a functional active ISC-machinery. Furthermore, Isa1 and Isa2 were found to interact with Grx5, the first ISC-component, which receives the de novo assembled [2Fe-2S]-cluster from Isu1 and transfers it to [2Fe-2S]-target proteins. The interaction with Isa1 and Isa2 suggests that Grx5 can also donate its Fe/S-cluster to the late acting ISC-components for conversion into a [4Fe-4S]-cluster. The Renilla Split-Luciferase assay revealed interactions between Yah1 and Isa1, Isa2 and Iba57. This data proposes that the process of Fe/S-cluster conversion depends on the supply with reducing equivalents. Because the Renilla Split-Luciferase assay displayed a concentration dependent assembly of the two Luciferase domains in mitochondria, the interactions described here have to be verified with an independent method.