Strukturelle und funktionelle Charakterisierung von Komponenten der eukaryotischen Eisen-Schwefel-Cluster-Biogenese-Maschinerie

Iron-sulfur clusters (Fe/S-clusters) are essential and versatile cofactors of numerous proteins and are present in virtually all living organism. Despite their relatively simple structure, their biosynthesis and assembly into apoproteins requires complex multi-protein biosynthesis-machineries which are conserved in eukaryotes. In the eukaryotic model organism S. cerevisiae the biogenesis of mitochondrial Fe/S-proteins depends on the mitochondrial ISC assembly system, whereas the synthesis of cytosolic and nuclear Fe/S-proteins additionally requires the mitochondrial ISC export machinery and the cytosolic CIA machinery. The biosynthesis of both mitochondrial and cytosolic nuclear Fe/S-proteins occurs in two major steps. First, an Fe/S-cluster is synthesized de novo on a scaffold protein. Second, the preassembled Fe/S-cluster is transferred and inserted in the target apoprotein. Whereas nearly all components of the biosynthesis machineries have been identified, the molecular mechanisms of Fe/S-cluster biosynthesis within the living cell are widely unknown. The de novo synthesis of Fe/S-clusters on the scaffold protein Isu1 of the mitochondrial ISC-assembly machinery requires an electron transfer by the NAD(P)H - ferredoxin reductase - ferredoxin chain. In the first part of this study it was shown that mitochondrial ferredoxins functionally and structurally are comprised of three distinct subclasses. Whereas members of the first subclass like human ferredoxin Fdx2 are specifically required for Fe/S-cluster biogenesis and heme A biosynthesis, ferredoxins of the second subclass such as the Fdx2-related human Fdx1 deliver electrons for steroid biosynthesis by cytochrome P450 enzymes (CYP). The third subclass is formed by the even more versatile ferredoxins from fungi including Yah1 from S. cerevisiae, which in addition to the functions of Fdx2, also plays an important role in the biosynthesis of coenzyme Q6. In this work, the three dimensional structure of human ferredoxin Fdx2 was determined at 1,7 Å resolution using X ray structure analysis. The folding of Fdx2 is virtually identical to that of Fdx1, despite the distinct substrate specificities of the two proteins. Structural differences were only observed in α-helix C and the region right after α-helix C. Genetic and biochemical experiments showed that the highly conserved C-terminus of Fdx2 is essential for its specific in vivo function in the biogenesis of Fe/S-clusters. An Fdx1 protein, which functions in Fe/S-cluster biogenesis, was successfully generated after exchange of the last 27 amino acids against the respective sequence from Fdx2. Further amino acid exchanges in the region of α-helix C and the loop covering the Fe/S-cluster increased the functionality showing the importance of these regions for substrate specificity. Vice versa, the generation of electron transfer capacity from Fdx2 to CYP was successful after introduction of the key mutation R73E. In this case, the gain of function was independent of the C terminus. The efficiency of specificity transfer was not complete; hence the key residue 73 is not solely responsible for the functional specificity. In fact, other global factors within the structure of the protein contribute to the specificity. It appears that the positively charged residue R73 in Fdx2 may inhibit transfer of electrons to CYP. Theoretical analysis of the dipole moment revealed an almost perpendicular orientation within the ferredoxins Fdx1 and Fdx2, despite their striking structural similarity. As the interaction of the highly charged ferredoxins with protein partners is based on charge-charge interactions, the difference in dipole moment orientation suggests an electrostatically driven steering mechanism during the encounter of the ferredoxins with their respective electron acceptors as a possible reason for their high degree of functional specificity. The steering effect might be a general mechanistic principle during the approach of proteins in electron transfer reactions. ..