Iron-sulphur clusters (Fe/S-clusters) are ubiquitous cofactors in many proteins. Their function ranges from electron transport via enzymatic activity to transcriptional regulation. Despite their universal importance and the ease of synthesis if model compounds starting from Fe(II), S2-, S8 and thioles, up to now the biosynthesis of this inorganic entities is not known. In Azotobacter a protein, namely NifS, was described which is essential for the assembly of the Fe/S-cluster in nitrogenase. NifS decomposes cysteine to alanine and sulphur, which is bound to a conserved cysteine as a persulfide. NifS-homologues were identified in a wide range of organisms from bacteria to mammals. Kessler and co-workers described C-DES from Synechocystis which shows sequence homology to NifS and which builds up the Fe/S-clusters in ferredoxin. This enzyme however produces pyruvate and lacks the essential cysteine. To gain insight into the mechanism of sulphur production, in this work NifS from A. vinelandii was cloned and purified. As no crystallisation condition for this protein was found, A NifS-homologue from T. maritima (tmNifS) was identified, cloned and purified. The structure of tmNifS was solved by multiple anomalous dispersion (MAD)-methods. In addition a substrate-complexed form of the enzyme was characterised. NifS is a homodimeric, PLP-dependent enzyme which belongs to the PLP-a-family. Therefore the structure of tmNifS is similar to other proteins from this family. The cysteine conserved among the NifS-like proteins is located in a highly flexible loop. It thereby can as well participate in the desulphuration reaction and also reach regions remote from the protein. This leads to the hypothesis that this loop can act as a 'molecular crane' and deliver the S0 produced directly to other proteins. UV-VIS spectroscopic measurements allow together with the active-site architecture to postulate a likely reaction mechanism, in which to a conserved histidine (H99) a crucial role is assigned. H99 stacks to the PLP-cofactor and thereby a mutual fine tuning of chemical properties of these two groups by means of electrostatic interaction seems likely. It was postulated that a protonated H99 stabilises the electron-rich chinonoid state of PLP, a deprotonated H99 on the other hand favours the electron-poor aromatic state. Using H99 as general acid/base, a reaction mechanism can be drawn which explains the desulphuration reaction. In addition the structure of C-DES as well as substrate- and product complexes thereof could be determined in collaboration with D. Kessler. C-DES uses another mechanism than NifS as it is lacking the conserved cysteine. It showed up that C-DES uses cystine as substrate and generates free rather than protein-bound cysteinepersulfide. The labile product is stabilised in a hydrophobic pocket where it is protected from hydrolysis. C-DES shows in contrast to NifS a significantly larger active-site cleft which makes a interaction which other proteins highly probable. It seems that - while NifS carries sulphur to other protein - in this case it seems to be necessary that other proteins take the sulphur from C-DES     «

Iron-sulphur clusters (Fe/S-clusters) are ubiquitous cofactors in many proteins. Their function ranges from electron transport via enzymatic activity to transcriptional regulation. Despite their universal importance and the ease of synthesis if model compounds starting from Fe(II), S2-, S8 and thioles, up to now the biosynthesis of this inorganic entities is not known. In Azotobacter a protein, namely NifS, was described which is essential for the assembly of the Fe/S-cluster in nitrogenase. Nif...     »