Molecular mechanisms of sulfur transfer during biosynthesis of iron-sulfur clusters in eukaryotes
Final Report Abstract
Virtually all known organisms depend on an ancient class of cofactors called iron sulfur (Fe/S) clusters which are essential for the function of enzymes in the mitochondria, the cytosol and the nucleus. In eukaryotic cells three proteinaceous machineries act together to accomplish the assembly of all Fe/S clusters. First, the ISC assembly machinery which is required for Fe/S protein maturation in the mitochondrial matrix and inner membrane. Second, the CIA machinery which assembles Fe/S clusters in the cytosol and nucleus. Even though several specialised CIA components work together to achieve cytosolic Fe/S protein maturation, this process depends also on the ISC assembly machinery. The connection between both pathways is established by the third important machinery, the mitochondrial ISC export apparatus. This set of three components is involved in the export of a yet unknown molecule from mitochondria which is strictly required for cytosolic Fe/S cluster maturation. This enigmatic substance (termed compound X) is known to represent an activated sulfur-containing species which can not be substituted by other cytosolic sulfur compounds. The goal of this project was the isolation and identification of compound X. Insights into the structural and chemical properties might eventually answer the question why sulfur can not be provided by the cytosol itself but instead nature retained the strict dependency of extramitochondrial Fe/S protein assembly on the mitochondrial ISC machineries. To gain insight into the mechanisms of compound X production and export, first an in vitro assay was established which is based on the de novo Fe/S cluster assembly on the cytosolic scaffold protein complex Cfd1/Nbp35 after export of compound X. Isolated intact mitochondria were functional in compound X production. The ability to act as a scaffold seems to be restricted to early CIA components as besides Cfd1/Nbp35 also Dre2 but not Nar1 or Leu1 supported this reaction, fully consistent with in vivo observations. To monitor Fe/S cluster assembly, either FeCl3 or 35S-cysteine was used as a marker. After isolation of the scaffold complex Cfd1/Nbp35 the quantification of associated radioactivity allowed conclusions on Fe/S cluster assembly. It was shown that this assembly strictly depends on the mitochondria-driven conversion of L-cysteine into some sort of activated sulfur species. This activity relies on an intact ISC and export machinery components, and on a membrane potential generated by a salt gradient under these anaerobic conditions. Staging experiments showed that only sulfur, but not iron is needed inside mitochondria to support the export reaction and yield reconstitution of the Fe/S cluster on Cfd1/Nbp35. Now that the in vitro conditions for compound X production were defined, this assay was used to accumulate compound X in the absence of the scaffold. The mitochondrial supernatant of such samples was subjected to different separation methods with the aim to isolate a fraction that was enriched in compound X. These fractions were then again used for Fe/S cluster reconstitution by adding iron and the scaffold complex. Anion exchange chromatography was successful in separating one fraction that was reconstitution active. Further analysis revealed that this fraction contained the unspecific by-product hydrogen sulfide. Due to the limited separation of this chromatography approach minor peaks were not resolved in the elution profile and thereby screening for low abundant sulfur compounds was not possible. To improve resolution reversed phase chromatography was performed using a C18 column. This separation method required a modification step to give the hydrophilic sulfur compounds a more hydrophobic character. This was achieved by labelling with monobromobimane which is a hydrophobic compound that reacts specifically with thiol groups. After labelling of the thio pool a good separation of all thiols could be obtained. Again it was observed that hydrogen sulfide is produced in significant amounts even in the absence of any reducing agents like DTT. Further experiments revealed that the presence of 1mM L-cysteine which was commonly used as a substrate was sufficient to reduce disulfide containing molecules like oxidised glutathione. As also compound X was expected to contain disulfide bridges this is a major problem during sample preparation which has to be solved in future studies. Taken together the results of this project provide a method for compound X production in vitro. We were successful in analysing the involvement of different mitochondrial enzymes in the process of compound X synthesis and export and scavenging by the CIA machinery. An efficient method was established to isolate the different thiolcompounds of the reaction mixture. Provided that the reducing conditions during sample preparation are eliminated or minimized, which is aspired by adjustment of L-cysteine concentrations and locking of compound X in a bound form, an established method set is now at hands for the eventual identification of compound X. This will then allow further mechanistic studies on the ISC export reaction.
