Chapter 1 in this thesis provides a short introduction to the biochemistry and chemistry of iron-sulphur systems, particularly in relation to biological nitrogen fixation and the inhibition of nitrogenases and hydrogenases by carbon monoxide. Chapter 2 describes some results bearing on the interaction of the inhibitor CO with simple iron-sulphur and iron-sulphur-vanadium systems under reducing conditions. It is shown that in the presence of CO an Fe454-centre can be reduced to carbonylated {Fe252} and {Fe35}-fragments. The crystal structure of [Fe35(CO)g ][PPh4]2 is described. Chapter 3 describes infra-red/electrolysis studies of the [Fe4S4 (5Ph)4 ]2-/CO system and of related Fe-S species. Evidence is presented for the occurrence of [Fe454(CO)12]2- as an intermediate in the pathway of reduction of [Fe454 (5Ph)4 ]2- to [Fe252 (CO)612- under an atmosphere of co. Chapter 4 describes the fortuitous isolation of [V(52)2(terpyridine)] from a reaction of 2,2',2"-terpyridine with [VFe3S4Cl3 (dmf)]- and its rational synthesis from 2,2' ,2"-terpyridine and [V54 ]3-. Some physical and chemical and electrochemical properties of this compound are described together with a description of its X-ray crystallographic structure.

Iron-sulfur clusters are protein cofactors that are critical for all life forms. Elaborate multi-component systems have evolved for the biosynthesis of these cofactors to protect organisms from the toxic effects of free iron and sulfide ions. In eukaryotes, the Fe-S cluster assembly machinery operates in the matrix space of the mitochondria and contains a myriad of proteins that mediate sulfur, iron, and electron transfer to assemble Fe-S clusters on the scaffold protein ISCU2 and then distribute these clusters to target proteins. Our lab has recently described stable 3, and 4-protein complexes composed of the cysteine desulfurase NFS1, the co-chaperone ISD11, and ISCU2 (SDU), and NFS1, ISD11, ISCU2, and FXN (SDUF) subunits. In the latter, SDUF, FXN functions as an allosteric activator switching this assembly complex on for Fe-S cluster biosynthesis. Insufficient expression of the mitochondrial protein FXN leads to a progressive neurodegenerative disease, Friedreich's Ataxia (FRDA). In ~2% of patients, FRDA is caused by one of 15 known missense mutations on one allele accompanied by the GAA repeat on the other leading to a complicated phenotype that includes loss of Fe-S clusters. Here we present in vitro evidence that FRDA FXN variants are deficient in their ability to bind the SDU complex, their ability to stimulate the sulfur transfer reaction from NFS1 to ISCU2, and in their ability to stimulate the rate of cluster assembly on ISCU2. Here, in vitro evidence is presented that FXN accelerates the sulfur transfer reaction from NFS1 to ISCU2. Additionally, we present kinetic evidence that identifies the most buried cysteine residue, C104 on ISCU2 as the sulfur acceptor residue suggesting, FXN stabilizes a conformational change to facilitate sulfur delivery. Subsequent mutational studies suggest FXN binding to SDU results in a helix to coil transition in ISCU2 exposing C104 to accept the persulfide sulfur and thereby accelerating the rate of sulfur transfer. We further provide the first biochemical evidence that the persulfide transferred to ISCU2 from NFS1 is viable in Fe-S cluster formation. In contrast to human FXN, the Escherichia coli FXN homolog CyaY has been reported to inhibit Fe-S cluster biosynthesis. To resolve this discrepancy, a series of inter-species enzyme kinetic experiments were performed. Surprisingly, our results reveal that activation or inhibition by the frataxin homolog is determined by which cysteine desulfurase is present and not by the identity of the frataxin homolog. These data are consistent with a model in which the frataxin-less Fe-S assembly complex exists as a mixture of functional and nonfunctional states, which are stabilized by binding of frataxin homologs. Intriguingly, this appears to be an unusual example in which modifications to an enzyme during evolution inverts or reverses the mode of control imparted by a regulatory molecule.

Iron-sulfur [Fe-S] clusters are protein cofactors that facilitate various life-sustaining biological processes. Their in vivo assembly is accomplished by three different systems known to date. These are: the NIF system which provides [Fe-S] clusters for nitrogenase and other nitrogen-fixing proteins, the SUF system which is induced during conditions of oxidative stress and iron starvation in E. coli, and the ISC system which serves as the housekeeping assembly apparatus. The latter is the focus of this dissertation and includes the proteins IscR, IscS, IscU, IscA, HscB, HscA, Fdx, and IscX. IscU is purified in its cluster-less (apo) form, but can serve as a scaffold to assemble [Fe-S] clusters in vitro in the presence of excess iron and sulfide. To test the scaffold hypothesis and gain insight into the events that occur during [Fe-S] cluster assembly and delivery, we developed two methods that allow the isolation of IscU and other ISC proteins in vivo. In the first method, Azotobacter vinelandii IscU is isolated from its native host, whereas in the second, it is isolated recombinantly from E. coli using a vector that allows expression of the entire isc operon. We found that IscU exists in vivo in two forms: apo-IscU and [2Fe-2S]2+ cluster-loaded IscU which are believed to be conformationally distinct. Both transient and stable IscU-IscS complexes were identified, indicating that the two proteins interact in vivo in a manner that involves their association and dissociation. The [2Fe-2S]2+-IscU species was present as a single entity, whereas significant amounts of apo-IscU were found associated with IscS, suggesting that IscU-IscS dissociation is triggered by the completion of [2Fe-2S] clusters. Both apo and [2Fe-2S]2+-IscU were predominantly monomeric whereas IscU-IscS complexes were determined to have an α2β2 composition. IscU was purified in the absence of the chaperones HscA and HscB and was also shown to accommodate a [2Fe-2S]2+ cluster similar to the one bound to IscU isolated from wild type cells. The findings suggest that [2Fe-2S]2+-IscU exists in one conformation in vivo and that any conformational changes on IscU are exerted after [2Fe-2S] cluster formation. In silico studies showed that a flexible loop containing the conserved LPPVK motif, which is responsible for interactions with HscA, may facilitate cluster exposure to either mediate its delivery to acceptor proteins or participation in the construction of [4Fe-4S] clusters. Experiments with NfuA, a protein similar to the C-terminal domain of NifU, demonstrated that NfuA and similar proteins might serve as [Fe-S] cluster carriers to accomplish the efficient delivery of nascent cofactors to the various recipient proteins. / Ph. D.

O complexo R2TP está presente em eucariotos, de leveduras a humanos, e está envolvido no correto dobramento de outras proteínas e montagem de complexos multiproteicos. R2TP é formado pelas proteínas Rvb1, Rvb2, Tah1 e Pih1/Nop17 em levedura, e direciona as chaperonas à proteínas alvo durante a montagem dos complexos. Os clusters Fe/S são sintetizados nas mitocôndrias e posteriormente transferidos para o citoplasma. Dre2 é uma proteína que contém cluster Fe/S, e está envolvida na transferência desses clusterspara outras proteínas citoplasmáticas. Nosso laboratório identificou a interação entre a subunidade Nop17 do complexo R2TP e Dre2 pelo método de duplo-híbrido, mas o papel desta interação ainda não foi elucidado. O objetivo deste trabalho foi o de estudar o papel funcional da interação entre Dre2 e Nop17 e identificar seus domínios de interação. Nossos resultados mostram que a porção N-terminal de Nop17 interage com a porção C-terminal de Dre2 e esta interação é necessária para a manutenção dos níveis de Dre2 na célula, indicando que o complexo R2TP atue na montagem do complexo CIA, de proteínas citosólicas Fe/S, do qual Dre2 faz parte. Dre2 também afeta a estabilidade de Nop17, sugerindo que Dre2 possa transferir um clusterFe/S para Nop17. Os dados mostrados aqui, portanto, indicam que a interação Nop17-Dre2 seja mutuamente importante para a estabilidade das duas proteínas / The R2TP protein complex is present in eukaryotes from yeast to humans, and is involved in the correct assembly of other protein or ribonucleoprotein complexes. R2TP is formed by proteins Rvb1, Rvb2, Tah1 and Pih1/Nop17 in yeast, and directs chaperones to target proteins during complexes assembly. Fe/S clusters are synthesized in mitochondria and later transferred to the cytoplasm. Dre2 is a Fe/S cluster protein, involved in transferring of Fe/S clusters to cytoplasmic proteins. Our laboratory has identified the interaction between the R2TP subunit Nop17 and Dre2 in the two-hybrid system. The aim of this work was to study the functional role of the interaction between Dre2 and Nop17, and to identify their domains of interaction. The results show that the N-terminal portion of Nop17 interacts with the C-terminal region of Dre2, and that this interaction is necessary for maintaining the levels of Dre2 in the cell, which suggests that the R2TP complex affects the cytosolic iron-sulfur protein assembly complex (CIA), of which Dre2 is a subunit. Dre2 also affects Nop17 stability, suggesting that Dre2 may transfer a Fe/S cluster to Nop17. The data here indicate that the interaction Nop17-Dre2 is mutually important for these proteins stabilities.

This thesis focuses on iron sulfur (Fe-S) cluster biogenesis by the ISC machinery in the mitochondrion of Trypanosoma brucei. Most of proteins in the pathway show conserved functions, while some features are distinct from their counterparts in other organisms. We also show here the essentiality of the ISC machinery in bloodstream stage despite the fact that the parasites contain the rudimentary mitochondrion in this stage. The key player for the ISC export machinery, which is indispensable in the maturation of extra-mitochondrial Fe-S proteins, shows some extraordinary phenomena which may imply the moonlighting function of the protein. I also show preliminary data of an ongoing project concerning a putative heme transporter. The results indicate role in heme uptake of the protein, but further study is required to confirm the function of the protein.

Les centres fer-soufre (Fe-S) sont des cofacteurs protéiques essentiels qui participent à un nombre important de fonctions cellulaires allant du métabolisme de l’ADN à la respiration mitochondriale. L’assemblage des centres Fe-S et leur insertion dans des protéines acceptrices requièrent l’activité d’une machinerie protéique dédiée. Bien que les protéines de la biogenèse des centres Fe-S soient conservées, plusieurs aspects fonctionnels et mécanistiques restent inconnus. Notre travail de thèse a consisté à caractériser les protéines mammifères de type A, ISCA1 et ISCA2, qui sont impliquées dans la biogenèse mitochondriales des centres Fe-S. En utilisant une approche couplant l’immunoprécipitation avec une analyse protéomique par spectrométrie de masse, plusieurs interactions protéiques d’ISCA1 et ISCA2 ont pu être identifiées. En plus d’une interaction entre ISCA1 et ISCA2, nous avons ainsi montré l’existence d’interactions spécifiques à chacune de ces protéines. Une approche de knockdown dans la souris via l’injection de virus adéno-associés, a permis de montrer l’absence de redondance fonctionnelle entre ISCA1 et ISCA2 puisque seul ISCA1 se trouve être nécessaire dans la maturation d’une catégorie de protéines à centre Fe-S. / Iron-sulfur clusters (Fe-S) are essential cofactors involved in different cellular processes ranging from DNA metabolism to respiration. Assembly of Fe-S clusters and their insertion into acceptor proteins is performed by dedicated protein machineries. Despite the high conservation from bacteria to man, different functional and mechanistic aspects of the Fe-S biogenesis remain elusive. In the present work, the function of the two mammalian A-type proteins ISCA1 and ISCA2 that are implicated in Fe-S biogenesis was investigated in vivo. First, an extensive analysis coupling immunoprecipitations and mass spectrometry led to the identification of a direct binding between ISCA1 and ISCA2 as well as specific protein partners of each protein. Furthermore, knockdown experiments in the mouse using adeno-associated virus provided clear evidence of the non-redundant function of ISCA1 and ISCA2, since only ISCA1 was shown to be required for a specific subset of mitochondrial Fe-S proteins.

O complexo R2TP está presente em eucariotos, de leveduras a humanos, e está envolvido no correto dobramento de outras proteínas e montagem de complexos multiproteicos. R2TP é formado pelas proteínas Rvb1, Rvb2, Tah1 e Pih1/Nop17 em levedura, e direciona as chaperonas à proteínas alvo durante a montagem dos complexos. Os clusters Fe/S são sintetizados nas mitocôndrias e posteriormente transferidos para o citoplasma. Dre2 é uma proteína que contém cluster Fe/S, e está envolvida na transferência desses clusterspara outras proteínas citoplasmáticas. Nosso laboratório identificou a interação entre a subunidade Nop17 do complexo R2TP e Dre2 pelo método de duplo-híbrido, mas o papel desta interação ainda não foi elucidado. O objetivo deste trabalho foi o de estudar o papel funcional da interação entre Dre2 e Nop17 e identificar seus domínios de interação. Nossos resultados mostram que a porção N-terminal de Nop17 interage com a porção C-terminal de Dre2 e esta interação é necessária para a manutenção dos níveis de Dre2 na célula, indicando que o complexo R2TP atue na montagem do complexo CIA, de proteínas citosólicas Fe/S, do qual Dre2 faz parte. Dre2 também afeta a estabilidade de Nop17, sugerindo que Dre2 possa transferir um clusterFe/S para Nop17. Os dados mostrados aqui, portanto, indicam que a interação Nop17-Dre2 seja mutuamente importante para a estabilidade das duas proteínas / The R2TP protein complex is present in eukaryotes from yeast to humans, and is involved in the correct assembly of other protein or ribonucleoprotein complexes. R2TP is formed by proteins Rvb1, Rvb2, Tah1 and Pih1/Nop17 in yeast, and directs chaperones to target proteins during complexes assembly. Fe/S clusters are synthesized in mitochondria and later transferred to the cytoplasm. Dre2 is a Fe/S cluster protein, involved in transferring of Fe/S clusters to cytoplasmic proteins. Our laboratory has identified the interaction between the R2TP subunit Nop17 and Dre2 in the two-hybrid system. The aim of this work was to study the functional role of the interaction between Dre2 and Nop17, and to identify their domains of interaction. The results show that the N-terminal portion of Nop17 interacts with the C-terminal region of Dre2, and that this interaction is necessary for maintaining the levels of Dre2 in the cell, which suggests that the R2TP complex affects the cytosolic iron-sulfur protein assembly complex (CIA), of which Dre2 is a subunit. Dre2 also affects Nop17 stability, suggesting that Dre2 may transfer a Fe/S cluster to Nop17. The data here indicate that the interaction Nop17-Dre2 is mutually important for these proteins stabilities.

No description available.

Présente chez les mammifères, mitoNEET (mNT) est une protéine à centre Fe-S ancrée à la membrane externe de la mitochondrie. Cette protéine dimérique possède un centre [2Fe-2S] par monomère lié de façon atypique à la protéine par trois cystéines et une histidine. Notre équipe a auparavant montré l’implication de mNT dans une nouvelle voie de réparation du centre [4Fe-4S] de l’Iron Regulatory Protein-1 (IRP-1), régulateur majeur de l’homéostasie du fer intracellulaire, par transfert du centre Fe-S de mNT à l’IRP-1 à réparer. Au cours de ma thèse, je me suis focalisée sur la caractérisation in vitro de la réaction de transfert de centre Fe-S de mNT vers une protéine réceptrice modèle, l’apo-ferrédoxine d’E. coli. En combinant des approches de biochimie et biophysique (réalisées en collaboration) à l’aide de protéines purifiées, cette étude a permis de démontrer que mNT agit comme un interrupteur moléculaire : lorsque son centre Fe-S est réduit, la protéine est extrêmement stable et le centre ne peut être ni perdu ni transféré; une fois oxydé, il peut alors être transféré à une protéine réceptrice. La présence d’oxygène n’affecte pas cette réaction même s’il s’agit d’un déterminant majeur de la stabilité de la protéine. De plus, la vitesse de transfert du centre est très sensible au pH, ce qui fait de mNT un senseur de pH. Ces études ont aussi montré que mNT est extrêmement résistante à H2O2 en comparaison à d’autres protéines de transfert de centre Fe-S. J’ai également étudié l’interaction d’une molécule anti-oxydante, le resvératrol-3 sulfate, avec mNT. Pour finir, je me suis intéressée à l’effet du glutathion sur mNT. Acteur majeur de la régulation de l’homéostasie rédox, le glutathion existe sous deux formes: oxydée (GSSG) et réduite (GSH). J’ai alors constaté que le GSH déstabilise fortement mNT à certains pH et peut même se lier à cette protéine. La fonction thiol du GSH et la formation de radicaux sur cette dernière sont clairement impliquées dans la déstabilisation de mNT. / Present in mammals, mitoNEET (mNT) is an Fe-S protein anchored to the outer mitochondrial membrane. This dimeric protein contains a [2Fe-2S] per monomer with an atypical ligation involving three cysteines and one histidine. Previously, our team proposed that mNT is involved in a new pathway dedicated to the reparation of the oxidatively damaged [4Fe-4S] cluster of human iron-regulatory protein-1 (IRP-1)/cytosolic aconitase, a key player of the regulation of cellular iron homeostasis. This reparation occurs via Fe-S cluster transfer from mNT to IRP-1 to repair. In the course of my thesis, I focused on the characterization of cluster transfer reaction from mNT to a model receptor protein, the E. coli apo-ferredoxin. Using purified proteins and combining biochemical approaches with biophysical ones performed in colaboration, this study showed that mNT acts as a redox switch: when the Fe-S cluster is reduced, the protein is extremely stable and it cannot be lost or transferred; when it is oxidized, it can be transferred to a receptor protein. Dioxygen does not affect this transfer reaction whereas this is a major determinant of protein stability. The transfer speed is highly sensitive to pH. Thus, mNT seems to act also as a pH sensor. Moreover, this study shows that mNT is extremely resistant to H2O2 compared to other Fe-S cluster transfer proteins. I also looked at the interaction of an antioxidant molecule, the resveratrol-3-sulfate, with mNT. Finally, I studied the effects of glutathione on mNT. Major player of the regulation of redox homeostasis, glutathione exists under two states: a reduced state (GSH) and an oxidized one (GSSG). I observed that GSH strongly destabilizes mNT at specific pHs and can even directly interact with the protein. The thiol function of GSH and the radical formation on this function are clearly involved in the mNT Fe-S destabilization.

This thesis highlights a divergent mitochondrial intermembrane assembly pathway in the parasitic protist Trypanosoma brucei. A comparative genomic study reveals the connection of Erv1 with the cytosolic iron-sulfur protein assembly (CIA) pathway in trypanosomatids. Further, the CIA machinery of T. brucei has been described using RNAi interference and other biochemical and complementation assays. Finally, part of the divergent CIA machinery has been identified in the human intestinal pathogen Giardia intestinalis by means of complementation assays in T. brucei.