Dissertationen zum Thema „Cluster [Fe-S]“

Many enzymes contain iron- sulfur (Fe-S) clusters which have a huge impact on their catalytic properties. These clusters may form part of the active site or form an electron relay system from the surface of the protein to the active site. Protein film electrochemistry (PFE) was utilized to elucidate the properties of some Fe-S cluster enzymes, namely, Hyd-1(a hydrogenase with an Fe-S electron relay), PceA (a reductive dehalogenase containing Fe-S clusters to facilitate electron transfer with redox partner) and CODH I_Ch and CODH II_Ch (carbon monoxide dehydrogenases with Fe-S electron relay systems and Ni-incorporated Fe-S clusters as active sites). The role of a proline residue at the active site in Hyd-1 was investigated and it was concluded that some local instability and adverse effect on H₂ activation were introduced upon replacement of proline with an alanine residue. The PceA dehalogenase was studied with PFE in terms of their interactions with various substrates and inhibitors. Furthermore, a method for performing 'film correction' for liquid substrates as that of the dehalogenase was established. Aspects of the catalytic cycle and effects of oxygen (O₂), peroxide (H₂O₂) and hydroxylamine (NH₂OH), a nitrogen-containing peroxide analogue on CODH ICh and CODH IICh were investigated with PFE. Finally, Electrochemical Impedance Spectroscopy (EIS), a technique involving application of alternating current (AC), was added to the portfolio was PFE techniques to compare CpI and CrHydA1 (hydrogenases with and without Fe-S electron relay system, respectively) in terms of time-dependent and time-independent processes within them. A novel term, exchange catalytic rate, for expressing inherent proficiency of the enzyme at zero-current potential was proposed and quantified. A means for measuring electroactive coverage and theoretical turnover during catalysis in PFE experiments was developed.

Dysfunctions in Fe-S protein biogenesis and mitochondrial iron accumulation in heart and neurones are part of the phenotype of a genetic neurodegenerative disease called Friedreich's ataxia. This pathology is caused by the deficiency of a mitochondrial protein, frataxin, highly conserved throughout species and currently thought to be a regulator of Fe-S cluster biosynthesis. The study of the mechanism of Fe-S cluster assembly in mitochondria is important to provide insights and valuable information potentially relevant for the study of iron-storage diseases. The biogenesis of iron sulfur clusters involves a complex molecular machine with macromolecular structures containing multiple subunits with specific functions. The high level of conservation of the components suggests the bacterial system as excellent model because of its inherent lower complexity. Isc is one of the operons that encodes proteins responsible for Fe-S cluster biogenesis in bacteria, including the desulfurase IscS, the scaffold protein IscU on which the Fe-S cluster is assembled, the two chaperones HscA and HscB, the trascription regulator IscR, a ferredoxin and two other proteins called IscA and YfhJ, whose role is still unclear. The function of the chaperones HscA and HscB is thought to assist the transfer of the cluster from the scaffold protein to the final acceptors. The main objective of this project was to get new evidence to understand the functions of the chaperones and the mechanisms by which they are involved in Fe-S cluster biogenesis and regulation through the application of structural biology and biochemistry. In particular, I focused on the structural and functional characterization of co-chaperone HscB and the analysis of its interactions with other members of the machinery through NMR and other biophysical techniques. My main findings are that HscB has an unprecedently reported interaction with IscS and that this interaction slows down cluster formation explaining a large plethora of evidence. These findings provide an entirely new perspective to the comprehension of the role of HscB and propose this protein as partner of central components of the Isc machine.

The organization of metabolism is an essential feature of cellular biochemistry. Metabolism does not occur as a linear assembly of freely diffusing enzymes, but as a complex web in which multiple interactions are possible. Because of the crowded environment of the cell, there must be structured and ordered mechanisms that control metabolic pathways. The following work will examine two metabolic pathways, one that is ubiquitous among living organisms and another that is entirely unique to plants, and examine the organization of each in an attempt to further define mechanisms that are fundamental features of metabolic control. One study offers some of the first characterizations of genes involved in [Fe-S] cluster assembly in Arabidopsis. The other explores the mechanisms that control localization of an enzyme that is part of the well-characterized flavonoid biosynthetic pathway. These two distinct pathways serve as unique models for genetic and biochemical studies that contribute to our overall understanding of plant metabolism.
Ph. D.

Thesis (Ph. D.)--Ohio State University, 2004.
Document formatted into pages. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2005 Aug. 18.

A system was developed for the controlled expression of genes in Azotobacter vinelandii by using genomic fusions to the sucrose catabolic regulon. This system was used for the functional analysis of the A. vinelandii isc genes, whose products are involved in the maturation of [Fe-S] proteins. For this analysis the scrX gene, contained within the sucrose catabolic regulon, was replaced by the A. vinelandii iscS, iscU, iscA, hscB, hscA, fdx, iscX gene cluster, resulting in duplicate genomic copies of these genes, one whose expression is directed by the normal isc regulatory elements (Pisc) and the other whose expression is directed by the scrX promoter (PscrX). Functional analysis of [Fe-S] protein maturation components was achieved by placing a mutation within a particular Pisc-controlled gene with subsequent repression of the corresponding PscrX-controlled component by growth on glucose as the carbon source. This experimental strategy was used to show that IscS, IscU, HscBA and Fdx are essential in A. vinelandii and that their depletion results in a deficiency in the maturation of aconitase, an enzyme that requires a [4Fe-4S] cluster for its catalytic activity. Depletion of IscA results in null growth only when cells are cultured under conditions of elevated oxygen, marking the first null phenotype associated with the loss of a bacterial IscA-type protein. Furthermore, the null growth phenotype of cells depleted for HscBA could be partially reversed by culturing cells under conditions of low oxygen. These results are interpreted to indicate that HscBA and IscA could have functions related to the protection or repair of the primary IscS/IscU machinery when grown under aerobic conditions. Conserved amino acid residues within IscS, IscU, and IscA that are essential for their respective functions and/or display a partial or complete dominant-negative growth phenotype were also identified using this system. Inactivation of the IscR repressor protein resulted in a slow growth phenotype that could be specifically attributed to the elevated expression of an intact [Fe-S] cluster biosynthetic system. This system was also used to investigate the extent to which the two [Fe-S] biosynthetic systems in A. vinelandii, Nif and Isc, can perform overlapping functions. Under normal laboratory growth conditions, no cross-talk between the two systems could be detected. However, elevated expression of Isc components as a consequence of inactivation of the IscR repressor protein results in a modest ability of the Isc [Fe-S] protein maturation components to replace the function of Nif-specific [Fe-S] protein maturation components. Similarly, when expressed at very high levels the Nif-specific [Fe-S] protein maturation components could functionally replace the Isc components. Oxygen levels were also found to affect the ability of the Nif and Isc systems to perform common functions. Nevertheless, the lack of significant reciprocal cross-talk between the Nif and Isc systems when they are produced only at levels necessary to satisfy their respective physiological functions, indicates a high level of target specificity with respect to [Fe-S] protein maturation.
Ph. D.

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

Strikt anaerobe Bakterien wie Clostridium difficile und C. scatologenes verwenden GRE, um die chemisch ungünstige Decarboxylierung von 4-Hydroxyphenylacetat zu p-Cresol zu katalysieren. Das Enzymsystem besteht aus einer Decarboxylase und dem zugehörigen Aktivierungsenzym. Die 4-Hydroxyphenylacetat-Decarboxylase (4Hpad) besitzt zusätzlich zum Protein-basierten Glycinradikal eine weitere Untereinheit mit bis zu zwei [4Fe-4S] Clustern und repräsentiert hierdurch eine neue Klasse von Fe/S-Cluster-haltigen GREs, die aromatische Verbindungen umsetzen. Das Aktivierungsenzym (4Hpad-AE) weicht vom Standardtypus ab, indem es zusätzlich zum S-Adenosylmethionin(SAM)-bindenden [4Fe-4S]-Cluster (RS-Cluster) mindestens einen weiteren [4Fe-4S]-Cluster bindet. In dieser Studie wurden heterologe Expressions- und Reinigungsprotokolle für 4Hpad und 4Hpad-AE entwickelt. Kristallstrukturen von 4Hpad cokristallisiert mit den Substraten (4-Hydroxyphenylacetat, 3,4-Dihydroxyphenylacetat) und dem Inhibitor (4-Hydroxyphenylacetamid) zeigten geringe strukturelle Änderungen im aktiven Zentrum des Proteins. Die Radikalbildung am 4Hpad-AE wurde durch die Überprüfung einer klassischen reduktiven Spaltung von SAM zu den Reaktionsprodukten 5’-Deoxyadenosin und Methionin bestätigt. EPR- und Mössbauer-Spektroskopische Analysen zeigten, dass 4Hpad-AE mindestens einen zusätzlichen [4Fe-4S] Cluster neben dem einzelnen RS-Cluster enthält. Die katalytische Notwendigkeit eines zusätzlichen Clusters wurde durch eine Mutationsanalyse untersucht, wobei eine verkürzte Version des Enzyms ohne die zusätzliche Cystein-reiche Insertion konstruiert wurde. Das verkürzte Mutante ohne die Bindungsmotive für die zusätzlichen Cluster gekennzeichnet, die Konfiguration, Stöchiometrie und die Funktion der zusätzlichen Cluster diagnostizieren.
4-hydroxyphenylacetate decarboxylase (4Hpad) is a two [4Fe-4S] cluster containing glycyl radical enzyme proposed to use a glycyl/thiyl radical dyad to catalyze the last step of tyrosine fermentation in Clostridium difficile and C. scatologenes by a Kolbe-type decarboxylation. The decarboxylation product p-cresol is a virulence factor of the human pathogen C. difficile. The small subunit of 4Hpad may have a regulatory function with the Fe/S clusters involved in complex formation and radical dissipation in the absence of substrate. The respective activating enzyme (4Hpad-AE) has one or two [4Fe-4S] cluster(s) in addition to the SAM-binding [4Fe-4S] cluster (RS cluster). The role of these auxiliary clusters is still under debate with proposed functions including structural integrity and conduit for electron transfer to the RS cluster. This study shows the optimized expression and purification protocols for the decarboxylase and the co-crystallization experiments and binding studies with 4-hydroxy-phenylacetate and 3,4-dihydroxyphenylacetate and with the inhibitor 4-hydroxy-phenylacetamide. The purification and characterization of active site mutants of decarboxylase are also done. Concerning 4-HPAD-AE, we report on the purification of code-optimized variants, and on spectroscopic and kinetic studies to characterize the respective i) SAM binding enthalpies, ii) rates for reductive cleavage of SAM and iii) putative functions of the additional Fe/S clusters. The truncated mutant lacking the binding motifs for the auxiliary clusters is characterized to diagnose the configuration, stoichiometry and function of the auxiliary clusters.

Hydrogenasen sind essentielle Enzyme im mikrobiellen H2-Kreislauf und werden als vielversprechende Katalysatoren in biologisch basierten H2-Technologien angesehen. Ein entscheidender Nachteil vieler Hydrogenasen ist ihre hohe O2-Sensitivität. Die membrangebundene Hydrogenase (MBH) aus Ralstonia eutropha ist eines der wenigen Beispiele für Hydrogenasen, die in Gegenwart von O2 katalytisch aktiv sind. Die molekularen Ursachen dieser O2 Toleranz sind bislang ungeklärt. In bisherigen Studien wurde lediglich das [NiFe]-Zentrum und dessen Umgebung auf Faktoren untersucht, die die O2-Toleranz des Enzyms hervorrufen könnten. In dieser Arbeit wurde daher der Fokus auf die kleine Untereinheit der MBH gelegt, in der sich drei elektronentransferierende Fe-S Cluster befinden. Die ligandierenden Aminosäuren dieser Fe-S Cluster wurden mittels ortsspezifischer Mutagenese verändert und die resultierenden MBH-Varianten physiologisch, biochemisch, spektroskopisch und elektrochemisch charakterisiert. Dabei wurde gezeigt, dass die O2-Toleranz der MBH maßgeblich auf einer Modifikation eines dieser drei Fe-S Cluster beruht. In der direkten Umgebung des zum aktiven Zentrum nächstgelegenen Fe-S Clusters befinden sich sechs statt vier Cysteine, wie in O2 sensitiven [NiFe]-Hydrogenasen. Die beiden zusätzlichen Cysteine um dieses proximale Cluster wurden gegen Glycine ausgetauscht, die an der entsprechenden Position in O2-sensitiven Hydrogenasen zu finden sind. Der Austausch der zusätzlichen Cysteine führte in vivo und in vitro zu einer erhöhten O2 Sensitivität der MBH. In EPR-spektroskopischen Untersuchungen wurde beobachtet, dass diese MBH-Variante veränderte elektronische Eigenschaften aufweist. Statt des für O2-tolerante Hydrogenasen typischen EPR-Spektrums wurde ein Signal detektiert, welches in O2-sensitiven Hydrogenasen zu finden ist. Anhand der Ergebnisse wurde ein Modell erstellt, das erklärt, wie eine modifizierte Fe-S Clusterkette zur O2-Toleranz von Hydrogenasen beiträgt.
Hydrogenases are essential for H2 cycling in microbial metabolism and serve as valuable blueprints for H2-based biotechnological application. Like many metalloproteins, most hydrogenases are extremely oxygen-sensitive and prone to inactivation by even traces of O2. The O2-tolerant membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha is one of the few examples that have established a mechanism enabling H2 uptake in the presence of ambient O2. The molecular mechanisms of this O2 tolerance are not yet unravelled. However, up to date, only the large subunit harbouring the [NiFe] active site has been in the focus of studies on O2 tolerance. In the present study, the role of the small subunit with its electron relay, consisting of three Fe-S clusters, was investigated. Amino acid residues involved in coordination of all three clusters were exchanged, and the resulting MBH variants were investigated with physiological, biochemical, electrochemical and spectroscopic methods. It is shown that the rare feature of O2 tolerance is crucially related to a modification of the electron transfer chain. The Fe-S cluster proximal to the catalytic centre is surrounded by six instead of the four conserved coordinating cysteines. Removal of the two additional cysteines renders the protein O2-sensitive in vivo and in vitro. Electron paramagnetic resonance spectroscopy of this MBH variant revealed a signal resembling the spectrum usually detected in O2-sensitive [NiFe]-hydrogenases. The data imply that the major mechanism of O2 tolerance is based on the reductive removal of oxygenic species guided by the unique architecture of the electron transport chain rather than a restricted access of O2 to the active site.

Cofatores ferro-enxofre são grupos prostéticos inorgânicos ubíquos e evolutivamente ancestrais, cuja formação é dependente de complexas maquinarias protéicas. Três sistemas de formação distintos já foram determinados, denominados sistemas NIF, ISC e SUF. Apesar de bem descritos em diversos organismos, estas maquinarias são pouco caracterizadas no filo Firmicutes, o qual agrupa diversas bactérias patogênicas, e onde Enterococcus faecalis aparece como um representante clinicamente relevante. O objetivo deste estudo foi identificar a maquinaria biossintética de formação dos cofatores [Fe-S] de E. faecalis mediante análises de bioinformática, determinação das regiões promotoras do operon e de elementos cis-atuantes, padrão de expressão gênica, caracterização bioquímica dos elementos encontrados e comparação entre as maquinarias de associação do cofator [Fe-S] presente em Proteobacteria e Firmicutes através da capacidade de complementação deste sistema nos sistemas ISC e SUF de Azotobacter vinelandii e Escherichia coli, respectivamente. Metodologias de bioinformática permitiram identificar representantes da maquinaria SUF de formação dos cofatores [Fe-S], previamente identificado em Proteobacteria, apresentando os genes sufB, sufC, sufD e sufS e a presença de sufU, o único representante homólogo do sistema ISC, codificando possível proteína arcabouço, no lugar de sufA; da mesma forma, sufE e sufR não foram identificadas. A alta conservação deste sistema foi verificada em Firmicutes através de análises filogenéticas. Análise de sequências primária e estrutural de SufU verificaram um padrão estrutural similar à IscU. Modelagem molecular de SufU de E. faecalis apresentou dados de alta flexibilidade na região do sítio ativo, bem como a presença de região específica em Firmicutes, denominada região Gram-positiva (GPR), possivelmente envolvida em interações com outros fatores e/ou reguladores. SufU e o complexo SufSU são capazes de reconstituir cofactor [4Fe-4S], apresentando-se portanto como a proteína arcabouço do sistema. A enzima SufS purificada apresenta PLP ligado como cofator e atividade de cisteína desulfurase. Esta enzima apresenta um residuo catalítico essencial de cisteína na posição 365 , e necessita SufU como ativador, onde outro residuo de cisteína (128) atua como aceptor do enxofre durante a reação de transpersulfuração. SufC apresenta atividade ATPase, porém em nível reduzido em comparação ao homólogo de E. coli; SufD apresenta alta similaridade com homólogo de proteobactérias. Por outro lado, SufB não apresenta os resíduos de cisteína previamente descritos como importantes na formação dos cofatores [Fe-S] em outros organismos, assim sua função no sistema ainda deve ser determinada. Experimentos in vivo demonstraram a conservação específica de sistemas biossintéticos dos cofatores [Fe-S], onde o operon SUF de E. faecalis não foi capaz de complementar os sistemas ISC de Proteobacteria, porém complementou sistema SUF de E. coli, tornando viáveis mutantes de ambos os operons sufABCDSE e iscRSU-hscBA-fdx.
Iron-sulfur clusters are ubiquitous and evolutionary ancient inorganic prosthetic groups, which biosynthesis depends on complex protein machineries. Three distinct assembly systems involved in the maturation of cellular Fe-S proteins have been determined, designated the NIF, ISC and SUF systems. Although well described in several organisms, these machineries are poorly understood in the Firmicutes phylum, which groups several pathological bacteria, where Enterococcus faecalis rises as a clinical relevant representative. The aim of this study was to identify the E. faecalis [Fe-S] cluster biosynthetic machinery through bioinformatics analysis, determination of operon promoter regions and cis-acting elements, relative genetic expression pattern, biochemical characterization of putative elements, and comparison of Proteobacteria and Firmicutes machineries through the ability of complementing Azotobacter vinelandii and Escherichia coli ISC and SUF systems, respectively. Bioinformatics methods enabled us to identify representatives of the SUF machinery for [Fe-S] cluster biosynthesis, previously verified in Proteobacteria showing conserved sufB, sufC, sufD and sufS genes and the presence of sufU, the only ISC homolog representative, coding for putative scaffold protein, instead of sufA; neither sufE nor sufR are present. High conservancy of this system for Firmicutes bacteria was verified through phylogenetic analysis. Primary sequences and structural analysis of the SufU protein demonstrated its structural-like pattern to the scaffold protein IscU. E. faecalis SufU molecular modeling showed high flexibility over the active site regions, and demonstrated the existence of a specific region in Firmicutes, the Gram positive region (GPR), a possible candidate for interaction with other factors and/or regulators. SufU is able to reconstitute a [4Fe-4S] cluster, such as the complex SufSU, arising as the scaffold protein in the system. Purified SufS corresponds to a PLP containing enzyme with cysteine desulfurase activity. It encloses a catalytically essential cysteine residue at position 365, and requires SufU as activator, where another cysteine residue (128) works as a proximal sulfur acceptor site for transpersulfurization reaction. SufC presents ATPase activity, though in a reduced level, when compared to the Escherichia coli homolog; SufD also shares high similarity with proteobacterial SufD. On the other hand, SufB does not present cysteine residues previously described as important involved in the [Fe-S] cluster formation process of other organisms, therefor its function in the system still have to be determined. In vivo experiments enabled us to dfemonstrate the conservancy of specific [Fe-S] cluster biosynthetic systems, where E. faecalis SUF operon was not able to complement Proteobacteria ISC systems, but complemented E. coli SUF system, turning viable mutants of both sufABCDSE and iscRSU-hscBA-fdx operons.

La biogènesi de centres Fe-S és necessària per múltiples processos cel·lulars. En l’estudi present s’ha demostrat que defectes en diferents etapes de la biosíntesi mitocondrial i citosòlica de centres Fe-S causen un increment en la mutagènesi espontània i hipersensibilitat a agents genotòxics, acompanyat d’un increment en la formació de foci associats a Rad52 i un augment en la fosforilació de la histona γ–H2A, tots aquests fets indicatius de la presència de lesions constitutives al DNA i d’inestabilitat genòmica. A més, d’un retard en la progressió a través de la fase S del cicle cel·lular i l’activació constitutiva del checkpoint de dany al DNA. Aquest darrer estimula l’augment en l’activitat de la ribonucleòtid reductasa mitjançant la disminució dels nivells de l’inhibidor Sml1 i la redistribució citosòlica de les subunitats enzimàtiques Rnr2/4. En paral·lel a la inestabilitat del genoma nuclear observada quan hi ha defectes en les diferents etapes de la maquinària ISC, s’ha demostrat també en aquest estudi una pèrdua constitutiva del DNA mitocondrial, i conseqüentment, defectes en la respiració i altres disfuncions mitocondrials.
La biogénesis de centros Fe-S es necesaria en múltiples procesos celulares. En el presente estudio se ha demostrado que defectos en diferentes etapas de la biosíntesis mitocondrial y citosólica de centros Fe-S causan un incremento en la mutagénesis espontanea e hipersensibilidad a agentes genotóxicos, acompañado de un incremento en la formación de foci asociados a Rad52 y un aumento en la fosforilación de la histona γ-H2A, todos estos hechos indicativos de la presencia de lesiones constitutivas en el DNA y de inestabilidad genómica. Además, un retraso en la progresión a través de la fase S del ciclo celular y la activación constitutiva del checkpoint de daño al DNA. Este último estimula el aumento de la actividad ribonucleótido reductasa mediante la disminución de los niveles del inhibidor Sml1 y la redistribución citosólica de las subunidades enzimáticas Rnr2/4. En paralelo a la inestabilidad genómica nuclear observada cuando hay defectos en las diferentes etapas de la maquinaria ISC, en este estudio se ha demostrado también una perdida constitutiva del DNA mitocondrial, y consecuentemente, defectos en la respiración y otras disfunciones mitocondriales.
The Fe-S clusters biogenesis is required for multiple cellular processes. In this work we demonstrate that defects at different stages of mitochondrial and cytosolic Fe-S clusters biosynthesis cause an increase of spontaneous mutagenesis and hypersensitivity to genotoxic agents, accompanied by an increment in Rad52-associated DNA repair foci and a hyperphosphorylated state of γ–H2A histone, altogether supporting the presence of constitutive DNA lesions and genomic instability. Furthermore, a delay in the progression of S phase in the cell cycle and constitutive activation of the DNA damage checkpoint. This last one stimulates the upregulation of the ribonucleotide reductase activity by promoting reduction of the levels of Sml1 inhibitor, as well as the cytosolic redistribution of the Rnr2/4 enzyme subunits. In parallel to the nuclear genome instability displayed when different ISC machinery stages are impaired, we also demonstrate in this work constitutive loss of mitochondrial DNA, and consequently, respiration defects and other mitochondrial dysfunctions.

Studies of ribosomal reading frame maintenance are often based on frameshift mutation suppression experiments. In this thesis, suppression of a frameshift mutation in Salmonella enterica serovar Typhimurium by a tRNA and a ribosomal protein are described. The +1 frameshift mutation hisC3072 (that contains an extra G in a run of Gs) is corrected by mutations in the argU gene coding for the minor tRNAArgmnm5UCU. The altered tRNAArgmnm5UCU has a decreased stability and reduced aminoacylation due to changed secondary and/or tertiary structure. Protein sequencing revealed that during the translation of the GAA-AGA frameshifting site the altered tRNAArgmnm5UCU reads the AGA codon inefficiently. This induces a ribosomal pause, allowing the tRNAGlumnm5s2UUC residing in the ribosomal P-site to slip forward one nucleotide. The same frameshift mutation (hisC3072) was also suppressed by defects in the large ribosomal subunit protein L9. Single base substitutions, truncations, and absence of this protein induced ribosome slippage. Mutated ribosome could shift to the overlapping codon in the +1 frame, or bypass to a codon further downstream in the +1 frame. The signal for stimulation of slippage and function of L9 needs to be investigated. During the search for suppressors of the hisD3749 frameshift mutation, a spontaneous mutant was isolated in the iscU gene that contained greatly decreased levels of the thiolated tRNA modifications ms2io6A and s2C. The iscU gene belongs to the iscR-iscSUA-hscBA-fdx operon coding for proteins involved in the assembly of [Fe-S] clusters. As has been shown earlier, IscS influences the synthesis of all thiolated nucleosides in tRNA by mobilizing sulfur from cysteine. In this thesis, it is demonstrated that IscU, HscA, and Fdx proteins are required for the synthesis of the tRNA modifications ms2io6A and s2C but are dispensable for the synthesis of s4U and (c)mnm5s2U. Based on these results it is proposed that two distinct pathways exist in the formation of thiolated nucleosides in tRNA: one is an [Fe-S] cluster-dependent pathway for the synthesis of ms2io6A and s2C and the other is an [Fe-S] cluster-independent pathway for the synthesis of s4U and (c)mnm5s2U. MiaB is a [Fe-S] protein required for the introduction of sulfur in ms2io6A. TtcA is proposed to be involved in the synthesis of s2C. This protein contains a CXXC conserved motif essential for cytidine thiolation that, together with an additional CXXC motif in the C-terminus may serve as an [Fe-S] cluster ligation site.

L’atome de soufre est un élément abondant et indispensable à la vie. Il est présent dans une grande diversité de biomolécules contenant du soufre, tels que certains acides aminés essentiels - comme la cystéine et la méthionine, qui sont à la base de diverses voies métaboliques -, des thionucléosides des ARNs de transfert, et de certains cofacteurs indispensables à de nombreux processus biologiques, comme les centres fer-soufre [Fe-S]. Les centres [Fe-S] sont connus pour leur activité rédox et leur rôle dans des réactions de transferts d’électrons. Ils ont des fonctions cellulaires importantes dans la photosynthèse, la respiration et la régulation de la traduction génétique dans des conditions de stress. Ma thèse a consisté en l’étude de deux familles d’enzymes à centres [4Fe-4S] impliquées dans le métabolisme du soufre : plusieurs thiouridylases d'ARN de transfert (MnmA d’E. coli, ThiI de l’archée Methanococcus maripaludis), catalysant l’insertion d’un soufre dans les uridines d’ARNt, et une ThioUracile DéSulfidase (TudS) catalysant l’abstraction du soufre du thiouracile. En combinant diverses méthodes de caractérisation biochimique (tests d’activité in vitro, mutagénèse dirigée) et biophysiques (spectroscopies UV-visible, RPE, Mössbauer, cristallographie aux rayons X), nous avons pu démontrer la nature chimique et le rôle de cofacteur du centre [4Fe-4S] dans les réactions non rédox catalysées par ces métalloenzymes. L’identification d’un intermédiaire réactionnel [4Fe-5S] in crystallo, dans la structure de l’enzyme TudS, a permis de confirmer une nouvelle fonction des centres [4Fe-4S] dans la catalyse de réactions non-redox, précédemment proposée pour les thiouridylases d’ARN de transfert (TtuA) : le centre [4Fe-4S] étant ligandé par trois acides aminés seulement, le quatrième fer non coordonné jouerait le rôle d’acide Lewis en liant et activant l’atome de soufre du substrat (sulfure exogène ou thiouracile, respectivement) pour catalyser la réaction de thiolation (thiouridylases d’ARNt) ou déthiolation (TudS)
The sulfur atom is an abundant and essential element for life. It is present in a wide variety of sulfur-containing biomolecules, such as certain essential amino acids - the cysteine and the methionine, which are at the center of various metabolic pathways -, transfer RNA thionucleosides, and certain essential cofactors participating in many biological processes, such as iron-sulfur centers [Fe-S]. The [Fe-S] centers are known for their redox activity and their role in electron transfer reactions. They have important cellular functions in photosynthesis, respiration, and regulation of gene translation under stress conditions. My thesis consisted of the study of two families of enzymes with [4Fe-4S] centers involved in sulfur metabolism: several transfer RNA thiouridylases (MnmA from E. coli, ThiI from archaea Methanococcus maripaludis), catalyzing sulfur insertion into tRNA uridines, as well as a ThioUracil DeSulfidase (TudS) catalyzing sulfur abstraction from thiouracil. By combining various biochemical (in vitro activity tests, site-directed mutagenesis) and biophysical (UV-visible spectroscopy, EPR, Mössbauer, X-ray crystallography) characterization methods, we were able to demonstrate the chemical nature and the role of the [4Fe-4S] cluster in the non-redox reactions catalyzed by these metalloenzymes. Identifying a reaction intermediate [4Fe-5S] in crystal, in the structure of the enzyme TudS, has confirmed a new function of the [4Fe-4S] clusters in the catalysis of non-redox reactions, previously proposed for transfer RNA thiouridylases (TtuA): the [4Fe-4S] cluster being liganded by only three amino acids, the fourth uncoordinated iron would play the role of Lewis acid by binding and activating the sulfur atom of the substrate (exogenous sulfide or thiouracil, respectively) to catalyze the reaction of thiolation (tRNA thiouridylases) or dethiolation (TudS)

Les interaccions antigen/anticòs són un dels tipus més interessants d’interaccions proteiques. La millor manera de prevenir les malalties causades per patògens és mitjançant l’ús de vacunes. L’aparició de la genòmica permet fer cerques a tot el genoma de nous candidats vacunals, tècnica anomenada vaccinologia inversa. L’estratègia més comuna on s’aplica la vaccinologia inversa és al disseny de vacunes de subunitats recombinants, que en general generen resposta immune humoral a causa de la presència d’epítops B en les proteïnes del patogen. Un problema important d’aquesta estratègia és la identificació de les proteïnes immunogèniques protectives del surfoma del patogen. El mimetisme epitòpic pot donar lloc a fenòmens autoimmunes relacionats amb diverses malalties humanes. El Capítol I d’aquesta tesi descriu una anàlisi computacional basat en la seqüència on, mitjançant l’aplicació de l’algorisme BLASTP, es van comparar bases de dades d’epítops B lineals coneguts i també de seqüències de proteïnes de superfície dels principals patògens bacterians respiratoris humans amb el proteoma humà. Es va trobar que cap dels 7353 epítops B lineals analitzats tenien regions d’identitat de seqüència amb proteïnes humanes capaces de generar anticossos i alhora que només l'1% de les 2175 proteïnes analitzades contenien alguna zona de seqüència compartida amb el proteoma humà. Aquestes troballes suggereixen l’existència d’un mecanisme per evitar l’autoimmunitat. També proposem una estratègia per corroborar o advertint sobre la viabilitat d’una proteïna que contingui un cert epítop B lineal de ser un bon candidat vacunal mitjançant estudis de vaccinologia inversa. En resum, els epítops sense cap tipus d’identitat de seqüència amb proteïnes humanes han de ser bons candidats vacunals, i al l’inrevés. El docking proteic és un mètode computacional per predir la millor manera en què interactuen les proteïnes, però, és possible identificar quina és la millor solució d’un programa de docking? La resposta habitual a aquesta pregunta és la solució que tingui més alta puntuació als outputs dels programes de docking, però les interaccions entre proteïnes són processos dinàmics, i moltes vegades la regió d’interacció és prou àmplia com per permetre diferents orientacions i/o energies d'interacció entre elles. En alguns casos, com en un multímer, es poden donar diverses regions d’interacció entre els monòmers. Aquests processos dinàmics impliquen interaccions, amb desplaçaments de superfície entre proteïnes, que porten a assolir la configuració funcional del complex proteic. Així doncs, en molts casos no hi ha una solució estàtica i única per a la interacció entre proteïnes, sinó que es donen diverses configuracions que també haurien de ser analitzades perquè podrien ser importants. Per extreure el conjunt de solucions més representatives dels outputs dels programes de docking, al Capítol II d’aquesta tesi es detalla el desenvolupament d’una aplicació de clústering no supervisada i automàtica, anomenada DockAnalyse. Aquesta aplicació es basa en el mètode ja existent de clústering DBscan, mitjançant el qual es busquen continuïtats entre els clústers generats per la representació de les dades dels outputs de docking. El mètode de clústering DBSCAN és molt robust i resol alguns dels problemes d’inconsistència dels mètodes clàssics de clústering com el tractament dels valors atípics i la dependència alhora de definir prèviament el nombre de clústers. Mitjançant representacions gràfiques i molt visuals, DockAnalyse fa que la interpretació de les solucions de docking sigui més fàcil permetent-nos trobar les més representatives. S’ha utilitzat aquesta nova aplicació per analitzar diverses interaccions proteiques i així poder modelar el comportament dinàmic de la interacció entre les proteïnes d’un complex. DockAnalyse també pot fer-se servir per a descriure regions d’interacció entre proteïnes i, per tant, orientar en futurs assajos de docking flexibles. L’aplicació (feta amb el paquet R) és oberta i accessible. La construcció dels Clústers Ferro-Sofre (ISC) en eucariotes implica interaccions entre diferents proteïnes, entre els quals es troba la proteïna Frataxina. Dèficits d'aquesta proteïna s'han associat amb excés de ferro dins del mitocondri i alteracions en la biogènesi dels ISC ja que es proposa que Frataxina actua com a donadora de ferro per a la construcció d'aquests ISC en aquest orgànul. Una reducció dràstica de Frataxina causa l'Atàxia de Friedreich, una malaltia neurodegenerativa hereditària humana que afecta principalment l'equilibri, la coordinació, els músculs i el cor. Aquest síndrome és l'atàxia autosòmica recessiva més comuna. Entre els mecanismes moleculars d' humans i de llevat que involucren Frataxina s'han trobat moltes similituds així que els llevats representen un bon model per a estudiar aquest procés. En llevat, el complex proteic que forma la plataforma central de muntatge dels passos inicials de la biogènesi dels ISC està composta per la Frataxina homòloga de llevat, el dímer Nfs1-Isd11 i la proteïna Isu. En general, està acceptat que la funció de les proteïnes implica interaccions amb altres proteïnes associades, però en aquest cas no se sap prou sobre l'estructura del complex de proteïnes i, per tant, com funciona exactament. En el Capítol III d'aquesta tesi es proposa un model del complex proteic necessari per a la biogènesi dels ISC amb el que es pretén aprofundir en detalls estructurals que expliquin la funció biològica. Per aconseguir aquest objectiu s'han utilitzat diverses eines de la bioinformàtiques, així com tècniques de modelització i programes de docking de proteïnes. Com a resultat, s'ha modelat l'estructura d'aquest complex proteic i també s'ha suggerit el comportament dinàmic dels seus components, juntament amb la dels àtoms de ferro i sofre necessaris per a la formació dels ISC. Aquestes hipòtesis podrien ajudar a comprendre millor la funció i les propietats moleculars de la proteïna Frataxina, així com els de les seves companyes presents al complex proteic.
Antigen/antibody interactions are one of the most interesting kinds of protein interactions. The best way to prevent diseases caused by pathogens is by the use of vaccines. The advent of genomics enables genome-wide searches of new vaccine candidates, called reverse vaccinology. The most common strategy to apply reverse vaccinology is by designing subunit recombinant vaccines, which usually generate humoral immune response due to B-cell epitopes in proteins. A major problem for this strategy is the identification of protective immunogenic proteins from the surfome of the pathogen. Epitope mimicry may lead to auto-immune condition related to several human diseases. Chapter I of this thesis describes a sequence-based computational analysis that was carried out applying the BLASTP algorithm where databases containing the known linear B-cell epitopes and the surface-protein sequences of the main human respiratory bacterial pathogens were compared to the human proteome. We found that none of the 7353 linear B-cell epitopes analyzed share any sequence identity region with human proteins capable of generating antibodies, and that only 1% of the 2175 exposed proteins analyzed contain a stretch of shared sequence with the human proteome. These findings suggest the existence of a mechanism to avoid autoimmunity. We also propose a strategy for corroborating or warning about the viability of a protein linear B-cell epitope to be a putative vaccine candidate in reverse vaccinology studies. Therefore, epitopes without any sequence identity with human proteins should be good vaccine candidates, and the other way around. Protein docking is a computational method to predict the best way by which proteins interact, but, is it possible to identify what the best solution of a docking program is? The usual answer to this question is the highest score solution, but interactions between proteins are dynamic processes, and many times the interaction regions are wide enough to permit protein-protein interactions with different orientations and/or interaction energies. In some cases, as in a multimeric protein complex, several interaction regions are possible among the monomers. These dynamic processes involve interactions with surface displacements between the proteins to finally achieve the functional configuration of the protein complex. Consequently, there is not a static and single solution for the interaction between proteins, but there are several important configurations that also have to be analyzed. To extract those representative solutions from the docking output datafile, Chapter II of this thesis details the development of an unsupervised and automatic clustering application, named DockAnalyse. This application is based on the already existing DBscan clustering method, which searches for continuities among the clusters generated by the docking output data representation. The DBscan clustering method is very robust and, moreover, solves some of the inconsistency problems of the classical clustering methods like, for example, the treatment of outliers and the dependence of the previously defined number of clusters. DockAnalyse makes the interpretation of the docking solutions through graphical and visual representations easier by guiding the user to find the representative solutions. We have applied our new approach to analyze several protein interactions and model the dynamic protein interaction behavior of a protein complex. DockAnalyse might also be used to describe interaction regions between proteins and, therefore, guide future flexible dockings. The application (implemented in the R package) is accessible. The assembly of Iron-Sulfur Clusters (ISCs) in eukaryotes involves interactions between different proteins, among which is important the protein Frataxin. Deficits in this protein have been associated with iron inside the mitochondria and impaired ISC biogenesis as it is postulated to act as the iron donor for ISCs assembly in this organelle. A pronounced lack of Frataxin causes Friedreich's Ataxia, which is a human neurodegenerative and hereditary disease mainly affecting the equilibrium, coordination, muscles and heart. Moreover, it is the most common autosomal recessive ataxia. High similarities between the human and yeast molecular mechanisms that involve Frataxin have been suggested making yeast a good model to study that process. In yeast, the protein complex that forms the central assembly platform for the initial step of ISC biogenesis is composed by yeast Frataxin homolog, Nfs1-Isd11 and Isu. In general, it is commonly accepted that protein function involves interaction with other protein partners, but in this case not enough is known about the structure of the protein complex and, therefore, how it exactly functions. In Chapter III of this thesis a model of the ISC biogenesis protein complex was proposed in order to gain insight into structural details that could end up with its biological function. To achieve this goal several bioinformatics tools, modeling techniques and protein docking programs were used. As a result, the structure of the protein complex and the dynamic behavior of its components, along with that of the iron and sulfur atoms required for the ISC assembly, were modeled. This hypothesis might help to better understand the function and molecular properties of Frataxin as well as those of its ISC assembly protein partners.

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

Ce travail de thèse propose de réaliser un nanofil électrique auto-assemblé constitué de protéines. L'unité de base de ce nanofil est une protéine chimère comprenant un domaine capable de former des fibres amyloïdes (Het-s 218-289) et un domaine capable d'effectuer des transferts d'électrons (une rubrédoxine). Le premier domaine permet la réalisation d'une fibre par auto-assemblage tandis que le deuxième est exposé à la surface de cette structure. Les caractéristiques redox du domaine exposé permettent aux électrons de se déplacer d'un bout à l'autre de la fibre par sauts successifs. Un tel nanofil a été créé et caractérisé par différentes techniques biophysiques. Ensuite, la preuve de la conduction des nanofils a été apportée sur des ensembles d'objets, de manière indirecte par électrochimie, et de manière directe par des mesures tension/courant. Ces travaux ouvrent la voie à la réalisation d'objets biocompatibles, biodégradables, possédant des propriétés électroniques exploitables dans des dispositifs technologiques.

De nombreux processus cellulaires tels que la respiration, la photosynthèse, l'assimilation du soufre et de l'azote ou encore la synthèse de vitamines ou de métabolites secondaires, dépendent de protéines à centre fer-soufre (Fe-S). Chez les plantes, la maturation de protéines Fe-S chloroplastiques, mitochondriales et cytosoliques ou nucléaires repose sur les machineries SUF, ISC et CIA respectivement. Dans le chloroplaste, un centre Fe-S, assemblé sur un complexe d'échafaudage SUFBC2D, est transféré via un ensemble de protéines de transfert vers les protéines cibles. L’objectif était de comprendre le rôle des protéines de transfert SUFA1, IBA57.2 et NFU1 en analysant leur capacité à lier des centres Fe-S et en isolant leurs partenaires au sein de la machinerie SUF et parmi les cibles chloroplastiques connues afin d’élucider les mécanismes moléculaires de ces interactions. Les expériences de reconstitution de centres Fe-S in vitro à partir des protéines recombinantes ont montré que SUFA1, NFU1 et le complexe SUFA1-IBA57.2 lient différents types de centres Fe-S. De plus, NFU1 incorpore un centre [4Fe-4S] au sein d’un dimère qui peut être transféré vers SUFA1, mais aussi vers les protéines cibles ISPG et THIC, deux enzymes impliquées dans la synthèse des isoprénoïdes et de la thiamine. L’ensemble des interactions identifiées par double hybride en levure et/ou BiFC pour ces protéines, mais aussi pour NFU2, NFU3 et HCF101, ont permis d’affiner leurs rôles respectifs dans la maturation des quelques 50 protéines Fe-S chloroplastiques
Numerous cellular processes such as respiration, photosynthesis, nitrogen and sulfur assimilation or vitamin and secondary metabolite synthesis, rely on iron-sulfur (Fe-S) proteins. In plants, the maturation of chloroplastic, mitochondrial and cytosolic or nuclear proteins depends on the SUF, ISC and CIA machineries respectively. In plastids, an Fe-S cluster is assembled on the SUFBC2D scaffold complex before being transferred to target proteins via transfer proteins. The objective was to understand the role of the SUFA1, IBA57.2 and NFU1 transfer proteins by analyzing their ability to bind Fe-S clusters and isolating their partners within the SUF machinery and among known chloroplastic targets in order to elucidate the molecular mechanisms of these interactions. In vitro Fe-S cluster reconstitution experiments using recombinant proteins have shown that SUFA1, NFU1 and the SUFA1-IBA57.2 complex bind different types of Fe-S clusters. In addition, NFU1 binds a [4Fe-4S] cluster within a dimer that can be transferred to SUFA1, but also to the target proteins, ISPG and THIC, two enzymes involved in the synthesis of isoprenoids and thiamine. All the interactions identified by yeast two-hybrid and/or BiFC for these proteins, but also for NFU2, NFU3 and HCF101, allowed refining their respective roles for the maturation of some of the 50 chloroplastic Fe-S proteins

Les composés soufrés, tels que la cystéine et certains cofacteurs, jouent un rôle essentiel dans les processus cellulaires. Cette thèse se concentre sur deux enzymes dépendantes d’un centre [4Fe-4S], impliquées dans le métabolisme du soufre chez l'archée anaérobie Methanococcus maripaludis : MmCyuA, une L-cystéine désulfidase, et MmLarE, une sulfurtransférase dépendante de l'ATP. La première partie porte sur MmCyuA, qui catalyse la conversion de la L-cystéine en sulfure d’hydrogène et 2-aminoacrylate, ultérieurement transformé en pyruvate et ammoniac. Les structures cristallographiques de MmCyuA, que nous avons obtenues, seule et en présence de l'inhibiteur sérine ou du produit pyruvate, sont les premières structures d'une cystéine désulfidase. Ces structures ainsi que nos résultats biochimiques et analyses spectroscopiques révèlent l’aptitude de MmCyuA à lier un cluster [4Fe-4S], indispensable à l’activité catalytique, via trois ou quatre cystéines. La structure de l'enzyme en complexe avec la sérine mime l'étape initiale de la réaction et suggère un mécanisme de désulfuration de la cystéine impliquant la formation d'un intermédiaire [4Fe-5S]. Des expériences comparatives de croissance de la souche sauvage de M. maripaludis et de la souche mutante dépourvue de l'enzyme MmCyuA soulignent l’importance de MmCyuA pour une croissance cellulaire optimale et pour permettre une croissance où la cystéine est utilisée comme unique source de soufre. Nous proposons que MmCyuA puisse transférer le sulfure lié au cluster à des accepteurs en aval des voies de biosynthèse des composés soufrés, tels que les enzymes de thiolation dépendantes d’un centre [4Fe-4S]. La deuxième partie concerne la structure et le mécanisme de MmLarE. Cette enzyme catalyse la conversion séquentielle des deux groupes carboxylates du précurseur du cofacteur de la lactate racémase en thiocarboxylates. Deux classes d’enzymes LarE existent, qui utilisent un mécanisme sacrificiel où une cystéine sert de source de soufre ou un mécanisme dépendant d’un cluster [4Fe-4S]. Nous rapportons la première structure cristallographique d'une enzyme LarE [4Fe-4S]-dépendante, sous ses formes apo (sans cluster) et holo (avec cluster). La structure de holo-MmLarE montre un cluster [4Fe- 4S], coordonné par trois cystéines seulement, avec le quatrième atome de fer lié à un ligand anionique (chlorure ou groupement phosphate). Ces structures, appuyées par nos études spectroscopiques, nous permettent de proposer un mécanisme dans lequel le cluster [4Fe-4S] lie un hydrogénosulfure, formant un intermédiaire [4Fe-5S]. Ce processus est similaire à celui des enzymes de thiolation de l'ARNt dépendantes d’un cluster [4Fe-4S]
Sulfur-containing compounds, such as cysteine and certain cofactors, play crucial roles in cellular processes. This thesis explores the sulfur metabolism in the anaerobic archaeum Methanococcus maripaludis, focusing on two [4Fe-4S]-dependent enzymes: L-cysteine desulfidase MmCyuA and ATP-dependent sulfur insertase MmLarE. The first part focuses on MmCyuA, which catalyzes the decomposition of L-cysteine into hydrogenosulfide and 2-aminoacrylate, subsequently converted into pyruvate and ammonia. The crystal structures of MmCyuA that we obtained, alone and in the presence of the serine inhibitor or the pyruvate product, are the first structures of a cysteine desulfidase. These structures, together with our biochemical results and spectroscopic analysis, reveal the capacity of MmCyuA to bind a [4Fe-4S] cluster, required for activity, using three or four cysteines. The structure of the enzyme in complex with serine mimics the initial step of the reaction and suggest a desulfuration mechanism for cysteine that involves the formation of a [4Fe-5S] intermediate. Comparative growth experiments between wild-type and CyuAdeficient M. maripaludis strains highlight the important role of MmCyuA for optimal growth and to enables growth using cysteine as the sole sulfur source. We propose that MmCyuA could transfer the cluster-bound sulfide to downstream acceptors, along the biosynthetic pathways of sulfurated compounds, such as [4Fe-4S]-dependent thiolation enzymes. The second part details the structure and mechanism of MmLarE. This enzyme catalyzes the sequential conversion of the two carboxylate groups of the precursor of the lactate racemase cofactor into thiocarboxylates. Two classes of LarE enzymes exist, using a sacrificial mechanism, in which a cysteine serves as the sulfur source, or a [4Fe-4S] cluster-dependent mechanism. We present the first crystal structure of a [4Fe-4S]-dependent LarE enzyme, in both its apo (without cluster) and holo (with cluster) forms. The crystal structure of holo-MmLarE reveals a [4Fe-4S] cluster coordinated by three cysteines only, with the fourth iron atom bound to an anionic ligand (chloride or phosphate group). These structures, along with our spectroscopic studies, support a mechanism in which the [4Fe-4S] cluster binds a hydrogenosulfide ligand, forming a [4Fe-5S]

L’ataxie de Friedreich est une maladie neurodégénérative sévère causée par un défaut d’expression de la frataxine (FXN), une petite protéine mitochondriale impliquée dans la biogenèse des centres fer-soufre (Fe/S), des groupement prosthétiques aux fonctions cellulaires essentielles. Chez les mammifères, il a été montré que la frataxine stimule la synthèse in vitro de centres Fe/S sur la protéine d’échaffaudage ISCU, grâce à l’augmentation de la production d’ions sulfures par le complexe NFS1-ISD11-ISCU. Cependant, le mécanisme par lequel la frataxine active la biogenèse des centres Fe/S n’a pas encore été défini. Nous avons étudié les effets de FXN sur les cinétiques de formation et de réduction des persulfures, des intermédiaires clés de la production d’ions sulfures, générés par la cystéiene désulfurase NFS1, à l’aide d’un test de détection des persulfures basé sur l’utilisation de composés synthétiques peptide-maléimide et de la spectrométrie de masse. Nous avons montré que FXN active deux réactions très similaires : la réduction du persulfure de NFS1 par des réducteurs à thiols comme le DTT, la L-cystéine et le glutathion et le transfert de soufre de NFS1 vers ISCU, conduisant à l’accumulation de persulfure sur la cystéine C104 d’ISCU. Nous avons constaté que la vitesse de réduction du persulfure d’ISCU par les thiols n’est pas affectée en présence de FXN et que ce persulfure est réduit plus lentement que celui de NFS1. Nous avons corrélé l’activation par FXN de la réduction du persulfure de NFS1 par les thiols à une stimulation de l’assemblage d’un centre Fe/S sur ISCU. Dans nos conditions expérimentales, l’atome de soufre du persulfure d’ISCU n’est pas incorporé dans le centre Fe/S synthétisé, mais nos résultats ne permettent pas d’exclure que ce persulfure puisse être réduit par une réductase dédiée, encore non identifiée. L’ensemble de nos données indiquent que le rôle de la frataxine est de contrôler la réduction du persulfure de NFS1, en augmentant les vitesses de transfert de soufre vers ISCU et de réduction du persulfure de NFS1 par les thiols
Friedreich ataxia is a severe neurodegenerative disease caused by reduced expression of frataxin (FXN), a small mitochondrial protein involved in iron-sulfur (Fe/S) cluster biogenesis which are prostetic groups with essential cellular functions. It has been shown in vitro that mammalian FXN activates Fe/S cluster synthesis on the scaffold protein ISCU, by rising up suflide ion production by NFS1-ISD11-ISCU complex. However, the mechanism by which frataxin stimulates Fe/S cluster biogenesis has not been yet defined. We have studied the effect of FXN on the kinetics of formation and reduction of persulfides that are key intermediates of sulfide ion production generated by NFS1, using mass spectrometry and a new detection assay for persulfide based on gel-mobility shift following alkylation by maleimide-peptide compounds. We demonstrate that frataxin activates two similar reactions : sulfur transfer from cysteine desulfurase NFS1 to ISCU leading to accumulation of a persulfide on ISCUcysteine C104 and reduction of NFS1 persulfide by thiol reducers such as DTT, L-cysteine and glutathion. We have observed that FXN does not stimulate the rate of ISCU persulfide reduction by thiols and that this persulfide is reduced much more slowly than NFS1 persulfide. We have then correlated the reduction of NFS1 persulfide with Fe/S cluster assembly. Under our experimental conditions, the sulfur from ISCU persulfide is not incorporated into the Fe/S cluster. However, we cannot exclude that an as yet not identfiied reductase could reduces ISCU persulfide and trigger Fe/S cluster assembly. Overall, our data point to a regulatory function of FXN as an enhancer of persulfide reduction, stimulating the rates of sulfur transfer to ISCU and NFS1 persulfide

Os cofatores de ferro-enxofre [Fe-S] estão entre os mais versáteis e antigos cofatores enzimáticos encontrados na natureza. As células têm explorado as propriedades eletrônicas e estruturais destes cofatores inorgânicos para uma ampla variedade de atividades incluindo a transferência de elétrons, a catálise e a ativação de substratos. Um grande número de proteínas está envolvido na biogênese dos cofatores [Fe-S], e este processo pode ser dividido em três etapas principais: (i) formação do enxofre elementar, (ii) montagem do cofator [Fe-S], e (iii) inserção do cofator em apoproteínas. As plantas realizam fotossíntese e respiração, dois processos que requerem proteínas Fe-S, sendo os únicos organismos em que a biossíntese destas proteínas é compartimentalizada. Diversos fatores afetam o desenvolvimento das plantas, entre eles, a temperatura baixa, fator limitante à produtividade e à distribuição geográfica das plantas, incluindo Eucalyptus grandis, uma espécie com grande importância econômica. Devido a esse fato, foi realizada uma análise transcricional dos genes codificados pelo sistema ISC de biossíntese de cofatores [Fe-S] NFS1, ISA1 e ISU1 de E. grandis por meio de PCR quantitativa (RT-qPCR), após plântulas desta espécie serem submetidas ao tratamento de frio. O gene NFS1 teve sua expressão reprimida nas primeiras 48 horas de tratamento, porém, após esse período observa-se um aumento da expressão gênica em relação à condição controle. O genes ISU1 e ISA1 apresentaram maior expressão gênica nas primeiras duas horas de tratamento, diminuindo drasticamente logo após este período. Foi verificado um aumento na quantidade relativa de Fe e S nos nas plântulas submetidas ao tratamento de frio, indicando um possível aumento na quantidade de cofatores [Fe-S] requeridos para o reestabelecimento da homeostase celular. As bactérias, por sua vez, desenvolveram pelo menos três sistemas de biossíntese, altamente conservados, que estão envolvidos na formação dos cofatores [Fe-S], sendo estes NIF, ISC e SUF. Em muitas proteobactérias, a regulação da produção de cofatores [Fe-S] pelos sistemas ISC e SUF é controlada por uma única proteína, IscR, pertencente à família de reguladores Rrf2. A proteína IscR possui um domínio de ligação ao DNA na região N-terminal e um segundo domínio de ligação de cofatores com três resíduos de cisteínas (Cys) altamente conservados. A ligação de um cofator do tipo [2Fe-2S] reprime a transcrição do seu próprio promotor in vitro. O genoma de Azotobacter vinelandii não inclui um sistema SUF completo e, portanto, permite o estudo dos efeitos da regulação de IscR não relacionada a SUF. No presente trabalho, objetivamos analisar a expressão do operon isc em linhagens selvagens e mutantes para IscR de A. vinelandii por meio das técnicas de sequenciamento do transcritoma e RT-qPCR. As substituições das Cys92, Cys104, His107 e a deleção de 120 pb da região codificadora do segundo domínio de IscR levaram à indução de um aumento da expressão de todo o operon isc. Notou-se também uma diferença fenotípica clara no tamanho das colônias portadoras das substituições de Cys e His, sendo estas menores em relação à linhagem selvagem. As substituições das Cys98 e Cys111, ou ainda a dupla substituição Cys98/111 não levaram a alteração da expressão do operon. A ligação ou não do cofator [Fe-S] é, portanto, responsável pela regulação do operon isc em A. vinelandii, bem como, de outros operons codificadores de proteínas envolvidas em cadeias tranportadoras de elétrons.
The iron-sulfur clusters [Fe-S] are among the oldest and most versatile enzyme cofactors found in nature. The cells have explored the structural and electronic properties of these inorganic clusters for a wide variety of activities including electron transfer, catalysis and activation of substrates. A large number of proteins is involved in the biogenesis of the [Fe-S] clusters, and this process can be divided into three main steps: (i) formation of elemental sulfur, (ii) assembly of the [Fe-S] cluster and (iii) insertion into apoproteins. Plants perform photosynthesis and respiration, two processes that require Fe-S protein, and in these organisms the synthesis of these proteins is compartmentalized. Several factors affect the development of plants, among them, the low temperature is a limiting factor to productivity and geographical distribution of plants, including Eucalyptus grandis, a specie with great economic importance. Due to this fact, we performed a transcriptional analysis by quantitative PCR (RT-qPCR) of the genes encoded by the E. grandis [Fe-S] cluster ISC system NFS1, ISA1 and ISU1 after seedlings were submitted to the chilling treatment. The NFS1 gene expression is repressed in the first 48 hours of treatment, but after this period there was an increase in gene expression relating to the control condition. The genes ISU1 and ISA1 showed higher gene expression in the first two hours of treatment, followed by a sharp decrease. There was an increase in the relative amount of Fe and S in the seedlings subjected to cold treatment, indicating a possible increase in the amount of [Fe-S] clusters, required for the reestablishment of cellular homeostasis. Bacteria have developed at least three synthesis systems, highly conserved, which are involved in the formation of Fe-S proteins, NIF, ISC and SUF. In many proteobacteria, the regulation of clusters production by ISC and SUF is controlled by a single protein, IscR, belonging to the Rrf2 regulators family. The protein IscR has a DNA binding site at the N-terminal domain and second cofactors binding domain with three cysteine residues (Cys) highly conserved. The binding of a [2Fe-2S] cluster represses the transcription of its own promoter in vitro. The genome of Azotobacter vinelandii does not include a full SUF system and thus permits the study of the effects of IscR regulation unrelated to SUF. In this study, the aim was to analyze the expression of isc operon in wild type and mutant strains of A. vinelandii IscR by the techniques of the transcriptome sequencing and qRT-PCR. The replacement of Cys92, Cys104, His107 and a deletion of 120 bp region encoding the second IscR domain led to an increased expression of the whole isc operon. It also showed a clear phenotypic difference in colonies size in the strains carrying the substitutions of His and Cys, it was smaller compared to the wild type strain. The replacement of Cys98 and Cys111, or the double substitution Cys98/111 not led to an altered operon expression. The [Fe-S] cluster binding or not, is therefore responsible for the regulation of the isc operon in A. vinelandii as well as of other operons encoding proteins involved in electron tranport chains.

La présence de cystéines réactives confère à de nombreuses protéines des propriétés redox et/ou la capacité à lier des ions métalliques. Ce projet, organisé en plusieurs axes, visait à caractériser des protéines possédant un motif CxxC conservé et éventuellement un repliement de type thiorédoxine (TRX) chez les plantes. Il s’avère que les TRX o1 et o2 mitochondriales, la protéine disulfure isomérase atypique PDI-A et la glutarédoxine (GRX) S16 chloroplastique d’A. thaliana exprimées sous formes recombinantes dans Escherichia coli incorporent toutes un centre Fe-S au sein d’homodimères dont la fonction reste à déterminer. L’analyse des propriétés redox des apo-protéines indiquent que la PDI–A et la GRXS16 ne possèdent pas ou peu d’activité oxydoréductase respectivement bien que des ponts disulfure intramoléculaires soient formés entre cystéines conservées. Dans le cas de la GRXS16, la régulation de son état redox se ferait via la lumière puisque le pont disulfure est réductible par des TRX mais pas par le glutathion. Le dernier axe de recherche concernait l’étude des propriétés de l’oxydoréductase MIA40 et de la flavine oxydase ERV1, impliquées dans l’import et le repliement oxydatif de protéines au sein de l’espace intermembranaire des mitochondries. Les résultats suggèrent que la singularité de ce système chez les plantes repose sur la structure atypique d’ERV1 et sa capacité à oxyder des protéines en présence de glutathion mais en absence de MIA40, qui est en revanche indispensable chez la levure ou l’homme
The presence of reactive cysteines confers redox properties and/or the ability to bind metal ions to numerous proteins. This project, organized in several axes, aimed at characterizing proteins with a conserved CxxC motif and possibly a thioredoxin (TRX) fold in plants. It appears that the mitochondrial TRX o1 and o2, the atypical protein disulfide isomerase PDI-A and the chloroplastic glutaredoxin (GRX) S16 from A. thaliana expressed as recombinant proteins in Escherichia coli all incorporated an Fe-S center within homodimers whose function remains to be determined. Analysis of the redox properties of apo-proteins indicates that PDI-A and GRXS16 have little or no oxidoreductase activity respectively although intramolecular disulfide bridges are formed between conserved cysteines. In the case of GRXS16, its redox state would be regulated by light as the disulfide bridge is reducible by TRX but not by glutathione. The last research axis concerned the study of the properties of the MIA40 oxidoreductase and the ERV1 flavine oxidase, involved in the import and oxidative folding of proteins within the inter-membrane space of mitochondria. The results suggest that the singularity of this system in plants is based on the atypical structure of ERV1 and its ability to oxidize proteins in the presence of glutathione but in the absence of MIA40, which is essential in yeast or humans

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.

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.

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.

SoxR est un régulateur transcriptionnel à centre [2Fe-2S] qui induit une réponse adaptative permettant à E. coli de résister aux composés redox actifs, générateurs de stress superoxyde. En présence de composés redox actifs, le centre [2Fe-2S] de SoxR est oxydé ce qui lui permet d’activer l’expression du gène soxS codant pour un régulateur transcriptionnel activant l’expression d’une centaine de gènes. Parmi les gènes du régulon SoxRS on trouve ceux permettant de résister au superoxyde mais aussi aux antibiotiques. J’ai montré qu’en présence de phénazine méthosulfate (PMS), un composé redox actif, la machinerie de biogénèse des centres Fe-S utilisée pour la maturation de SoxR est différente suivant les conditions environnementales. En effet, en aérobie la maturation de SoxR est assurée par la machinerie SUF, alors qu’en anaérobie c’est la machinerie ISC qui intervient. J’ai également étudié l’importance de SoxR, et des machineries ISC et SUF, dans la résistance aux antibiotiques induite par la présence de PMS. J’ai montré qu’en présence de PMS, E. coli peut résister à la norfloxacine, par un mécanisme SoxR dépendent, et ceci quelque soit la machinerie de biogénèse des centres FeS présente. D’autre part, j’ai étudié l’impact des conditions environnementales, comme la teneur en CO2 dans l’atmosphère sur la capacité d’ E. coli à résister au stress oxydant. J’ai testé, expérimentalement les prédictions obtenues par un modèle d’équations différentielles permettant de simuler la concentration des ROS dans la cellule. J’ai montré que le CO2 a un effet de protection lors d’un stress au H2O2 probablement en capturant les HO• produits par la réaction de Fenton
SoxR is a [2Fe-2S] cluster-containing transcriptional regulator that mounts the adaptive response allowing E. coli to tolerate superoxide-propagating compounds. When cells are exposed to redox cycling drugs the Fe-S cluster of SoxR undergoes a reversible univalent oxidation to yield the oxidized active protein. The only known target of SoxR is the soxS gene that is itself a transcriptional regulator activating the expression of more than 100 genes including those for superoxide and antibiotic resistance. I showed that the machinery used to mature SoxR under phenazine methosulfate (PMS) exposition, a redox cycling drug, was different depending on the environmental conditions used. In aerobiosis, the SUF machinery ensured SoxR maturation, while in anaerobiosis the ISC machinery was required. I also monitored the implication of SoxR, the ISC and SUF machineries, in antibiotic resistance induced by PMS exposition. I showed that E. coli can resist to norfloxacin under PMS exposition in a SoxR-dependent manner whatever the Fe-S cluster biogenesis machinery available. Last, I studied the impact of environmental conditions, such as atmospheric CO2 concentration, on the ability of E. coli to cope with oxidative stress. I have experimentally tested the predictions obtained by a mathematical model that simulates ROS dynamics. I showed that carbon dioxide has a protective effect on hydrogen peroxide stress likely by scavenging the radical hydroxyl produced by the Fenton reaction

Les protéines [Fe-S] sont des enzymes ubiquitaires, assurant des fonctions clés au sein des organismes vivants. La biosynthèse des centres [Fe-S], à savoir les processus permettant un assemblage correct des atomes de fer et de soufre au sein des protéines cibles, requièrent la participation de machineries protéiques complexes. Parmi elles se trouve la machinerie SUF qui intervient dans des conditions de stress oxydant et de carence en fer. Elle est composée de six gènes sufABCDSE. La protéine SufA est proposée comme étant une protéine scaffold ayant pour rôle de préassembler transitoirement des centres [Fe-S] et de les transférer à des protéines cibles. Elle possède trois résidus cystéines conservés proposés comme étant les ligands des centres [Fe-S]. SufA est obtenue principalement sous forme apo après purification. Le centre [Fe-S] peut être reconstitué chimiquement in vitro. Dans ces conditions, SufA contient un mélange de centres [2Fe-2S] et [4Fe-4S]. Nous avons alors isolé SufA native métallée après purification à partir de tout l’opéron suf en anaérobiose, et montré qu’elle contient un centre [Fe-S], plutôt de type [2Fe-2S], transférable efficacement à la ferrédoxine. Nous avons également étudié les mécanismes moléculaires de formation du cluster dans SufA. SufA est capable de fixer à la fois du soufre, au niveau de ses trois cystéines conservées, et du fer, majoritairement au niveau d’atomes d’azote et d’oxygène. Ces éléments sont mobilisables pour la formation d’un centre [Fe-S] en milieu réducteur. Enfin, des expériences préliminaires réalisées in vitro avec des mutants dirigés n’ont pas permis d’identifier la nature exacte des ligands du centre [Fe-S] dans SufA
[Fe-S] proteins are ubiquitous enzymes which play key roles within all living organisms. The biosynthetic process by which iron and sulfur atoms are combined in a controlled way into target proteins requires complex machineries. Among them, we can find the SUF machinery which is involved under oxidative stress and iron starvation conditions. This system is composed of six genes sufABCDSE. The SufA protein is described as a scaffold protein which is able to assemble transient [Fe-S] clusters and to transfer them to target apoproteins. Moreover, SufA contains three conserved cysteine residus which are proposed to be the ligands of the [Fe-S] clusters. SufA is purified mainly in apo form. The [Fe-S] cluster can be reconstituted chemically in vitro. Under these conditions, SufA contains a mix of [2Fe-2S] clusters and [4Fe-4S] clusters. We isolated the native SufA protein in a metalled form after purification from the suf operon in anaerobic conditions. We showed that SufA contains an [Fe-S] cluster, probably a [2Fe-2S] cluster, which can be transferred to the ferredoxine efficiently. We also studied the molecular mechanisms of the assembly of [Fe-S] cluster in SufA. SufA is able to bind both sulfur atoms, coordinated by the three conserved cysteines, and iron atoms, mainly coordinated by nitrogen and oxygen ligands. These two elements can be used for the assembly of [Fe-S] clusters in the presence of a reductant. Lastly, we carried out preliminary in vitro experiments with site-directed mutant proteins to determine the ligands of [Fe-S] clusters in SufA, but today, the nature of the ligands remains unclear

Les glutaredoxines (GRXs) són tiol oxidoreductases àmpliament repartides entre organismes procariotes i eucariotes. La Grx5 de llevat Saccharomyces cerevisiae es troba a la matriu mitocondrial i participa en la síntesi dels centres ferro-sofre (ISCs), que són co-factors necessaris per a diversos processos cel•lulars essencials des la respiració, la regulació de l'expressió gènica i el metabolisme de l'ADN-ARN. Algunes proteïnes amb ISCs estan involucrades en el metabolisme de l'ADN, més precisament en la replicació de l'ADN o en els processos de reparació del mateix. Treballs recents van demostrar que els defectes en el metabolisme de ISCs comprometen l'estabilitat del genoma cel•lular, i aquesta inestabilitat està associada a la predisposició a múltiples càncers humans. Mitjançant l'ús d'un mutant Δgrx5 de S. cerevisiae com a model d'estudi vam intentar entendre millor la relació entre la inestabilitat genòmica i els defectes en la biosíntesi de ISCs. L'absència de la proteïna mitocondrial Grx5 condueix a un augment de la inestabilitat genètica. Aquesta inestabilitat del genoma no depèn de l'acumulació de ferro que es produeix en les cèl • lules que no tenen Grx5. Les cèl • lules que no expressen Grx5 tenen majors nivells de dany constitutiu a l'ADN, que s'associa específicament a la formació de "focis" associats a Rad52. Les cèl • lules que no tenen Grx5 i Ssq1 són hipersensibles a agents que danyen l'ADN de forma additiva. Aquesta sensibilitat és independent de l'estat d'estrès oxidatiu constitutiu que es produeix en les cèl • lules que no tenen Grx5. L'absència de les proteïnes Grx5 i Ssq1 provoca un retard en la progressió del cicle cel • lular a través de la fase S. La via de reparació de l'ADN, recombinació homòloga podria tenir un paper important en la reparació dels danys en l'ADN en les cèl • lules del mutant Δgrx5.
Las glutaredoxinas (GRXs) son tiol oxidoreductasas ampliamente distribuídas entre organismos procariotas y eucariotas. La proteína Grx5 de levadura Saccharomyces cerevisiae se encuentra en la matriz mitocondrial y participa en la síntesis de los centros hierro-azufre (ISCs), que son co-factores necesarios para varios procesos celulares esenciales, tales como la respiración hasta la regulación de la expresión génica y el metabolismo del ADN-ARN. Algunas proteínas asociadas a ISCs están involucradas en el metabolismo del ADN, más precisamente en su replicación o en procesos de reparación del mismo. Trabajos recientes demostraron que los defectos en el metabolismo de ISCs comprometen la estabilidad del genoma celular, y esta inestabilidad está asociada a la predisposición a múltiples cánceres humanos. Mediante el uso de un mutante Δgrx5 de S. cerevisiae como modelo de estudio quisimos abordar la comprensión de la relación entre la inestabilidad genómica y los defectos en la biosíntesis de ISCs. La ausencia de la proteína mitocondrial Grx5 conduce a un aumento de la inestabilidad genética. Esta inestabilidad del genoma no depende de la acumulación de hierro que se produce en las células que carecen Grx5. Las células que no expresan Grx5 tienen mayores niveles de daño constitutivo al ADN, que se asocia específicamente a la formación de “focis” asociados a Rad52. Las células que carecen Grx5 y Ssq1 son hipersensibles a agentes que dañan el ADN de forma aditiva. Esta sensibilidad es independiente del estado de estrés oxidativo constitutivo que se produce en las células que carecen de Grx5. La ausencia de las proteínas Grx5 y Ssq1 provoca un retraso en la progresión del ciclo celular a través de la fase S. La vía de reparación del ADN, recombinación homóloga podría desempeñar un papel importante en la reparación de los daños en el ADN en las células del mutante Δgrx5.
Glutaredoxins (GRXs) are thiol oxidoreductases widely spread among prokaryotic and eukaryotic organisms. Saccharomyces cerevisiae Grx5 is located at the mitochondrial matrix and participates in the synthesis of iron-sulphur clusters (ISCs), which are co-factors required for several essential cellular processes, such as respiration, regulation of gene expression and DNA-RNA metabolism. Some ISC proteins are involved in DNA metabolism, more precisely in DNA replication and/or repair processes. Recent works reported that defects in ISC metabolism compromise the stability of the cellular genome, and this instability is associated to human predisposition towards multiple types of cancers. Using the yeast grx5 mutant as a model we wanted to gain further insight in the relationship between genomic instability and defects in ISC biosynthesis.The absence of mitochondrial protein Grx5 leads to an increase in genetic instability. This genomic instability is not dependent on the iron accumulation produced in cells lacking Grx5. The cells that do not express Grx5 have higher levels of constitutive DNA damage, specifically associated with the formation of "focis" related with Rad52. Cells lacking Ssq1 and Grx5 are hypersensitive to DNA-damaging agents in an additively way. This sensitivity is independent of the constitutive oxidative stress state that occurs in the cells depleted of Grx5. The absence of proteins Ssq1 and Grx5 causes a delay in cell cycle progression through S phase. The DNA repair pathway homologous recombination could play an important role in the repair of DNA damage in cells Δgrx5 mutant.

Die NAD+-reduzierende Hydrogenase aus Ralstonia eutropha (SH) katalysiert die reversible H2-Oxidation in Verbindung mit der Reduktion von NAD+ in Gegenwart von Sauerstoff. Die bemerkenswerte O2-Toleranz des Enzyms wurde zuvor auf eine für [NiFe]-Hydrogenasen ungewöhnliche Struktur des Wasserstoff-spaltenden Zentrums zurückgeführt. Diese Hypothese wurde in dieser Arbeit mittels in situ-Spektroskopie an SH-haltigen Zellen widerlegt. Um die folgende Untersuchung der aus sechs Untereinheiten und mindestens acht Kofaktoren bestehenden SH zu erleichtern, wurde das Enzym mittels genetischer Methoden in seine beiden Module aufgeteilt. Das die H2-Oxidation katalysierende Hydrogenase-Modul beinhaltete ein FMN-Molekül, welches für die reduktive Reaktivierung des oxidativ modifizierten Zentrums benötigt wird. Das Diaphorase-Modul besaß ebenfalls ein FMN, und die Reduktion von NAD+ wurde von der Anwesenheit von O2 nicht beeinträchtigt. Neben Wasserstoff reagierte das [NiFe]-Zentrum der SH auch mit Sauerstoff. Dabei wurde sowohl Wasserstoffperoxid- als auch Wasser im Hydrogenase-Modul freigesetzt. Die Sauerstofftoleranz der SH basiert auf einer kontinuierlichen Reaktivierung des durch Sauerstoff oxidierten [NiFe]-Zentrums. Aufgrund der außergewöhnlichen Sauerstofftoleranz stellt die SH ein vielversprechendes System für die wasserstoffgetriebene Regeneration von NADH in gekoppelten enzymatischen Reaktionen dar. In dieser Arbeit wurde ein SH-Derivat durch rationale Mutagenese konstruiert, das in der Lage war, ebenso den Kofaktor NADP+ wasserstoffabhängig zu reduzieren. Durch Ganzzellansätze kann die zeitaufwändige und kostenintensive Proteinreinigung vermieden werden. Um die wasserstoffabhängige in-vivo-Kofaktorregeneration zu ermöglichen, wurde die SH in Pseudomonas putida heterolog produziert. Die in dieser Arbeit erzielten Ergebnisse sind sowohl für das molekulare Verständnis der H2-abhängigen Katalyse als auch für die biotechnologische Anwendung der O2-toleranten SH relevant.
The NAD+ reducing hydrogenase from Ralstonia eutropha (SH) catalyzes the reversible oxidation of hydrogen in connection with the reduction of NAD+ in the presence of oxygen. The remarkable oxygen tolerance was previously related to an unusual [NiFe] active site with four instead of two cyanide ligands. This hypothesis was rejected in this study by using in situ spectroscopy on SH containing cells. To simplify the investigation of the six-subunit and at least eight cofactors containing SH, the enzyme was separated into its two modules by genetic methods. The hydrogen oxidizing hydrogenase module contained one FMN molecule, which was required for the reductive reactivation of the oxidatively modified active site. The diaphorase module carried a second FMN. The reduction of NAD+ was not affected by the presence of oxygen. In addition to hydrogen, the [NiFe] center of the SH reacted with oxygen. Both hydrogen peroxide and water were released by the hydrogenase module. The oxygen tolerance of the SH is based on a continuous reactivation of the oxidized [NiFe] center. Due to the oxygen tolerance, the SH is a promising system for hydrogen based NADH regeneration in coupled enzymatic reactions. In this study a SH derivative was constructed by means of rational mutagenesis. The SH derivative was able to reduce the cofactor NADP+ by hydrogen oxidation. The time consuming and costly protein purification can be avoided by using whole cell approaches. In order to allow the hydrogen dependent in vivo cofactor regeneration, SH was heterologously produced in Pseudomonas putida. The results obtained in this study are relevant for the molecular understanding of hydrogen dependent catalysis and for the biotechnological application of the oxygen tolerant SH.

Chez les plantes, les protéines à centre fer-soufre (Fe-S) sont impliquées dans de nombreux processus cellulaires (e.g. photosynthèse, respiration). La maturation de ces protéines nécessite la synthèse de novo des centres Fe-S à l’aide de machineries d’assemblage spécifiques. Les plantes possèdent trois machineries d’assemblage nommées SUF, ISC et CIA, dédiées à la maturation des protéines plastidiales, mitochondriales et nucléaires ou cytosoliques, respectivement. Lors de la maturation des protéines mitochondriales, un centre [2Fe-2S] est initialement assemblé sur la protéine d’échafaudage ISU puis transféré vers les apoprotéines cibles à l’aide de chaperons et de diverses protéines de transfert. Si ces étapes semblent suffisantes pour la maturation de protéines incorporant des centres [2Fe-2S], un couplage réductif de deux centres [2Fe-2S] est nécessaire pour la maturation des protéines de type [4Fe-4S]. Cette conversion nécessite des protéines de transfert et un donneur d’électrons, potentiellement la même ferrédoxine que celle qui agit déjà lors des étapes précoces pour la réduction du soufre. En combinant des approches moléculaires, biochimiques et génétiques, l’implication des protéines de transfert NFU et ISCA et des ferrédoxines mitochondriales (mFDX) dans les étapes tardives de transfert et de conversion a été explorée au cours de cette thèse chez la plante modèle Arabidopsis thaliana. Des expériences de complémentation en levure ont démontré que les protéines NFU et ISCA de plantes peuvent assurer les mêmes fonctions que leurs orthologues respectifs, suggérant que ces étapes tardives ont été conservées. Cependant, contrairement à la levure, l’analyse de lignées n’exprimant pas les deux protéines NFU indiquent qu’elles sont essentielles pour le développement de l’embryon. Au niveau moléculaire, les analyses effectuées à l’aide d’approches in vivo et/ou in vitro ont permis d’identifier une interaction entre ISCA1a ou ISCA1b et ISCA2, NFU4 et NFU5 mais aucune interaction avec les deux mFDX dont le rôle dans les dernières étapes d’assemblage des centres Fe-S reste donc incertain. La formation d’holo-hétérocomplexes entre ISCA1 et ISCA2 a été confirmée par co-expression chez E. coli et purification des protéines recombinantes. Globalement, en associant la littérature à propos de la machinerie ISC et les résultats obtenus, le modèle qui ressort est que des hétérocomplexes ISCA1/2 agiraient immédiatement en amont des protéines NFU qui permettraient a minima la maturation des centres [4Fe-4S] de la lipoate synthase. Ce seul partenaire pourrait expliquer en grande partie la létalité d’un mutant nfu4 x nfu5 car l’activité de plusieurs protéines centrales pour le métabolisme mitochondrial dépend de l’acide lipoïque
In plants, iron-sulfur (Fe-S) proteins are involved in crucial processes such as photosynthesis and respiration. The maturation of these proteins requires the de novo synthesis of their Fe-S clusters through dedicated assembly machineries. Plants have three Fe-S cluster assembly machineries, namely SUF, ISC and CIA, devoted to the maturation of plastidial, mitochondrial and nuclear or cytosolic proteins, respectively. During the mitochondrial Fe-S protein maturation, a [2Fe-2S] cluster is first assembled on the ISU scaffold protein then transferred to target proteins with the help of chaperones and various transfer proteins. If these steps are sufficient for the maturation of [2Fe-2S] proteins, a reductive coupling process of two [2Fe-2S] clusters is required for the maturation of [4Fe-4S] proteins. This conversion needs transfer proteins and an electrons donor, potentially the same ferredoxin which acts during the first step of the Fe-S cluster biogenesis for sulfur reduction. By combining molecular, biochemical and genetic approaches, the involvement of NFU and ISCA transfer protein and mitochondrial ferredoxin (mFDX) in the late transfer and conversion steps has been explored during this PhD project by using the Arabidopsis thaliana plant model. Yeast complementation experiments have demonstrated that plant NFU and ISCA proteins have functions similar to their respective orthologs, suggesting that these late steps are conserved. However, unlike yeast, the characterization of nfu mutant lines indicates that both proteins are essential for early embryonic development. At the molecular level, in vivo and in vitro approaches have shown an interaction between ISCA1a or ISCA1b and ISCA2, NFU4 and NFU5 but no interaction with the two mFDX whose participation in the late steps remains uncertain. The formation of ISCA1-ISCA2 holo-heterocomplexes has been confirmed by co-expression in E. coli and purification of recombinant proteins. Overall, the literature and results obtained here highlight a model where ISCA1/2 heterocomplexes would act immediately downstream of NFU proteins which would a minima allow [4Fe-4S] cluster maturation of the lipoate synthase. This sole partner could primarily explain the lethality of a nfu4 x nfu5 double mutant because the activity of several proteins central for the mitochondrial metabolism depends on lipoic acid

Les deplecions de DNA mitocondrial (mtDNA) i les alteracions de NFU1 són dos grups de malalties que afecten vies molt importants dins de la mitocòndria i que alteren el metabolisme energètic. Clínicament, els pacients afectes de depleció de mtDNA es caracteritzen per mostrar un ampli ventall de símptomes, depenent del gen afectat. Els símptomes clínics són molt greus i la majoria de vegades porten a la mort del pacient. Els pacients amb mutacions en el gen NFU1, descrits per primera vegada en aquest treball, presenten una clínica més homogènia, caracteritzada sobretot per presentar una encefalopatia infantil fatal i/o hipertensió pulmonar i un fenotip bioquímic molt particular però ben definit, caracteritzat principalment per presentar acidosi làctica, hiperglicinèmia i deficiència de l’activitat piruvat deshidrogenasa. L’objectiu principal d’aquesta tesis ha consistit en millorar i implementar metodologies per a contribuir al diagnòstic, i millorar la comprensió de la fisiopatologia d’aquestes deficiències. Per a tal fita s’ha posat a punt una tècnica de PCR a temps real per estudiar el número de còpies de mtDNA i poder relacionar-lo amb l’activitat citrat sintasa. A més, la depleció s’ha pogut estudiar en biòpsies incloses en parafina, una important font de material biològic arxivat mai utilitzat fins al moment. D’aquesta forma s’han pogut estudiat 50 pacients amb sospita clínica de depleció de mtDNA i cercar mutacions en els gens relacionats; DGUOK, MPV17, C10orf2, SUCLG1, SUCLA2 i POLG. L’estudi molecular ens ha permès identificar 4 mutacions no descrites prèviament, c.70+5G>A en el gen MPV17, c.1048G>A i c.1049G>T en el gen SUCLA2, i c.531+4A>T en el gen SUCLG1. A més s’han identificat 7 mutacions prèviament descrites en un total de 10 pacients (8 famílies). Quan va ser possible, es va quantificar el ràtio mtDNA/nDNA i la activitat citrat sintasa en la mateixa mostra de teixit, proveint noves dades per l’estudi de les MDS. La depleció relacionada amb la citrat sintasa, (mtDNA/nDNA)/CS, ha donat resposta a certes discrepàncies observades entre els resultats de depleció i els resultats de la cadena respiratòria mitocondrial en alguns pacients. Per altra banda, utilitzant el mapatge per homozigositat, es va identificar una mutació de canvi de sentit en homozigositat en el gen NFU1 (c.622G>T, p.Gly208Cys), que codifica per una proteïna altament conservada en totes les espècies i que està implicada en la biogènesis dels clústers de sulfur de ferro (Fe-S). Aquesta ha esta la primera vegada que s’ha associat mutacions en aquest gen a patologia humana. El fenotip bioquímic dels pacients suggeria una activitat deficient de l’enzima lipoic àcid sintasa (LAS), una proteïna que necessita clústers de Fe-S com a cofactor. Es va poder constatar una disminució del grau de lipoilació de les proteïnes dependents d’àcid lipoic, el que suggeria una manca d’ activitat LAS. Utilitzant models cel·lulars hem demostrat que la proteïna NFU1 és necessària com a donadora de sulfur per la biosíntesi d’àcid lipoic i també ens ha permès conèixer la funció específica en la biosíntesi dels clústers de Fe-S i en la maduració de proteïnes Fe-S, concretament LAS i la succinat deshidrogenasa (SDH). La descripció clínica, bioquímica y genètica d’aquesta malaltia és molt important pel diagnòstic de nous pacients i obre les portes a la cerca d’altres gens implicats en la biosíntesi de l’àcid lipoic, així com al disseny futur de noves estratègies terapèutiques.
Mitochondrial DNA (mtDNA) depletion syndromes (MDS) and NFU1 defects are two groups of diseases affecting crucial mitochondrial pathways of energetic metabolism. Clinically, patients affected of mtDNA depletion displayed a wide range of symptoms, depending on the altered gene. The clinical symptoms are severe and in most cases lead to death of the patient. Patients with NFU1 mutations, described for the first time in this paper, present a more homogeneous clinical phenotype, characterized by fatal infantile encephalopathy and / or pulmonary hypertension. NFU1 patients also showed a peculiar, but well defined, biochemical phenotype, presenting with lactic acidosis, hyperglycinemia and deficiency of pyruvate dehydrogenase activity. The main objective of the present thesis is to improve and to implement new methods for the diagnosis and understanding of the pathophysiology of these deficiencies. To this goal, a real-time PCR technique has been developed to study the mtDNA copy number and its relationship to citrate synthase activity in MDS patients. In addition, mtDNA depletion has been studied in formalin-fixed paraffin-embedded tissues, an important source of biological material never used for this purpose. We studied 50 paediatric individuals suspected to have mtDNA depletion and the appropriate MDS genes have been screened according to their clinical and biochemical phenotypes. Mutational study of DGUOK, MPV17, SUCLA2, SUCLG1 and POLG allowed us to identify 4 novel mutations; c.70+5G>A in MPV17, c.1048G>A and c.1049G>T in SUCLA2 and c.531+4A>T in SUCLG1, and 7 already known mutations in 10 patients (8 families). When possible, we quantified mtDNA/nDNA and CS activity in the same tissue sample, providing an additional tool for the study of MDS. The ratio (mtDNA/nDNA)/CS has shed some light in the discrepant results between the mtDNA copy number and the enzymatic respiratory chain activities of some cases. Using homozigosity mapping, we identified a homozygous missense mutation in NFU1 gene (c.622G> T, p.Gly208Cys), which encodes a conserved protein suggested to participate in Fe-S cluster biogenesis. This is the first time that a clinical phenotype has been associated with mutations to NFU1. The biochemical phenotype suggested an impaired activity of the Fe-S enzyme lipoic acid synthase (LAS), a protein that requires Fe-S cluster as a cofactor. Direct measurement of protein-bound lipoic acid in individual tissues indeed showed marked decreases, which suggested a lack of LAS activity. Human cell models studies showed that NFU1 protein is required as sulfur donor for the biosynthesis of lipoic acid and it performs a specific function in mitochondrial Fe-S proteins maturation, particularly succinate dehydrogenase and LAS (SDH). Clinical, biochemical and genetic description of NFU1 disease is very important for the diagnosis of new patients and will allow us to find other genes involved in the biosynthesis of lipoic acid, and provided the basis for the future design of new therapeutic strategies.

Les protéines à centre Fe-S sont impliquées dans de nombreux processus cellulaires. In vivo, la formation des centres Fe-S est réalisée par des machineries multi-protéiques dont ISC et SUF, conservées chez les eucaryotes et les procaryotes. D’autres composants participent à la formation des centres Fe-S chez les eucaryotes, comme la frataxine (FXN). La FXN est une protéine présente chez l’homme, les plantes, la levure ou encore les bactéries à Gram négatif. Chez les eucaryotes, l’absence de FXN conduit à des phénotypes drastiques comme une accumulation de fer dans la mitochondrie, une diminution drastique de l’activité d’enzymes à centre Fe-S ou encore des dommages oxydatifs. Chez l’homme, un déficit en FXN est responsable d’une maladie neurodégénérative, l’ataxie de Friedreich. A la différence des eucaryotes, chez les procaryotes comme Escherichia coli, l’absence de CyaY, homologue bactérien de la FXN, ne conduit à aucun des phénotypes évoqués ci-dessus.Durant ma thèse, je me suis intéressée au rôle de CyaY chez E. coli. J’ai montré que, in vivo, CyaY favorise la formation des centres Fe-S via la machinerie ISC. Un lien génétique entre CyaY et IscX a également pu être établi, montrant que ces deux protéines participent à la formation des centres Fe-S in vivo. Je me suis ensuite intéressée aux bases moléculaires pouvant expliquer la différence entre les phénotypes liés à l’absence de FXN chez les eucaryotes et les procaryotes. J’ai montré que le résidu 108 de IscU joue un rôle clé pour la dépendance de CyaY. Enfin, pour mieux comprendre le rôle de CyaY chez E. coli, j’ai réalisé une approche globale en caractérisant le transcriptome du mutant ∆cyaY
Fe-S cluster containing proteins are involved in many cellular processes such as respiration, DNA repair or gene regulation. In vivo, Fe-S cluster biogenesis is catalysed by specific protein machineries, ISC and SUF, conserved in both eukaryotes and prokaryotes. Frataxin (FXN) is a small protein found in humans, plants, yeast and Gram negative bacteria. In eukaryotes, a defect in FXN leads to drastic phenotypes such as mitochondrial iron accumulation, drastic decrease of Fe-S cluster protein activity, sensitivity to oxidants. In humans, FXN deficiency is responsible for the neurodegenerative disease, Friedreich’s ataxia. In prokaryotes like E. coli, a defect in CyaY, the bacterial FXN homolog, does not lead to significant phenotypes compared to the wild-type strain. During my thesis, I investigated the role of the bacterial FXN CyaY in E. coli. I showed that, in vivo, CyaY assisted the ISC-catalyzed Fe-S cluster biogenesis. A genetic link was also observed between cyaY and iscX, demonstrating that these proteins participate in Fe-S cluster biogenesis. In a second part, I investigated the differences between the impact of the eukaryotic versus prokaryotic FXN. I showed that the IscU 108th residue is crucial for the CyaY-dependency. Finally, I used a transcriptomic approach to test whether CyaY has a global role in E. coli

The proteins that bind Fe/S clusters are fundamental in numerous cellular processes, in all types of organisms, not only for their involvement in electron transport, but also in gene expression regulation, like sensors of environmental or intracellular conditions. Biogenesis of the Fe/S proteins is a complex process requiring a large number of accessory proteins. In this pathway, the [4Fe-4S] cluster formation and delivery remain not defined, but mutations in genes encoding proteins involved in ISC maturation, such as NFU1, BOLA3, ISCA2 and IBA57, have been related to a new group of diseases, the multiple mitochondrial dysfunction syndromes (MMDS). In particular, BOLA3 and NFU1 deficiency causes MMDS types 2 and 3, results in the reduced functionality of the respiratory complexes I and II as well as of lipoic acid-dependent enzymes. Similar clinical and biochemical phenotypes in patients with mutations in the late acting factor protein (NFU1), suggested a functional correlation between the two proteins in the late step of the mitochondrial Fe/S protein maturation, even if detailed biochemical investigations of their molecular role in mitochondria have not been reported yet. It’s known that the protein NFU1 binds a [4Fe-4S] cluster, although is still unknown how it is assembled on it. MMDS5 has recently been described in a clinical case report of patients carrying a mutation in ISCA1, but with no further analysis. In the first year of my PhD studies, I characterized the [4Fe-4S] NFU1 and how the [2Fe-2S] GLRX5/BOLA3 complex cooperates in the cluster transfer process. During this year I have worked on two new projects. The second project is focus on in vitro interaction and cluster transfer between NFU1 and ISCA1/ISCA2 complex. The current prevailing model, largely based on studies in S. cerevisiae, proposes that NFU1 receives a [4Fe-4S] cluster assembled on the ISCAs proteins system and then transfers it to selected apo target proteins with the assistance of BOLA3. Based on this model, the project addresses the possible molecular interactions between the [4Fe-4S] ISCA2 protein or/and the ISCA1/ISCA2 complex with NFU1, all involved in [4Fe-4S] cluster transfer to targets enzymes.

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.

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.

Mitochondrial dysfunction has been implicated for a wide range of human diseases. One of the major biosynthetic processes in human mitochondria is the biogenesis of Iron-Sulfur (Fe-S) clusters which primarily involves in electron transfer reactions during oxidative phosphorylation (OXPHOS). Defects in Fe-S cluster biogenesis process leads to mitochondrial dysfunction and that eventually results in various human mitochondrial disorders. One of the major mitochondrial disorders associated with Fe-S cluster biogenesis impairment is exercise intolerance disorder ISCU myopathy, which is a result of loss of function of Fe-S cluster scaffold protein ISCU. Our biochemical results using yeast model system and HeLa cells lines suggests that ISCU Myopathy results in defective Fe-S cluster biogenesis in mitochondrial compartment. As a result, electron transport chain (ETC) complexes demonstrate significant reduction in their redox properties, leading to loss of cellular respiration. Furthermore, in ISCU Myopathy, mitochondria display enhancement in iron levels and reactive oxygen species, thereby causing oxidative stress leading to impairment in the mitochondrial functions. On the other hand, in mammalian mitochondria, the initial step of Fe-S cluster assembly process is assisted by NFS1-ISD11 complex, which delivers sulfur to the scaffold protein ISCU during Fe-S cluster synthesis. In humans, loss of ISD11 function leads to development of respiratory distress disorder, Combined Oxidative Phosphorylation Deficiency 19 (COXPD19). Our study maps the important ISD11 amino acid residues critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these critical ISD11 residues to alanine leads to its compromised interaction with NFS1, which results in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Moreover, our findings highlight that, COXPD19 associated R68L ISD11 mutant displays reduced affinity to form a stable sub-complex with NFS1, thereby fails to prevent NFS1 aggregation, resulting impairment of Fe-S cluster biogenesis. The prime affected machinery is the ETC complex which demonstrates compromised redox properties, causing diminished mitochondrial respiration in COXPD19 patients. In summary, our findings provide compelling evidence that respiration defect due to impaired biogenesis of Fe-S clusters in ISCU myopathy patients, leads to manifestation of complex clinical symptoms. Additionally, our study highlights the role of ISD11 protein in Fe-S cluster biogenesis and maps the surface residues of ISD11 protein that are involved in interaction with sulfur donor protein NFS1. Moreover, we have demonstrated the molecular basis of disease progression of COXPD19 as a result of R68L ISD11 mutation.

Mitochondrial dysfunction has been implicated for a wide range of human diseases. One of the major biosynthetic processes in human mitochondria is the biogenesis of Iron-Sulfur (Fe-S) clusters which primarily involves in electron transfer reactions during oxidative phosphorylation (OXPHOS). Defects in Fe-S cluster biogenesis process leads to mitochondrial dysfunction and that eventually results in various human mitochondrial disorders. One of the major mitochondrial disorders associated with Fe-S cluster biogenesis impairment is exercise intolerance disorder ISCU myopathy, which is a result of loss of function of Fe-S cluster scaffold protein ISCU. Our biochemical results using yeast model system and HeLa cells lines suggests that ISCU Myopathy results in defective Fe-S cluster biogenesis in mitochondrial compartment. As a result, electron transport chain (ETC) complexes demonstrate significant reduction in their redox properties, leading to loss of cellular respiration. Furthermore, in ISCU Myopathy, mitochondria display enhancement in iron levels and reactive oxygen species, thereby causing oxidative stress leading to impairment in the mitochondrial functions. On the other hand, in mammalian mitochondria, the initial step of Fe-S cluster assembly process is assisted by NFS1-ISD11 complex, which delivers sulfur to the scaffold protein ISCU during Fe-S cluster synthesis. In humans, loss of ISD11 function leads to development of respiratory distress disorder, Combined Oxidative Phosphorylation Deficiency 19 (COXPD19). Our study maps the important ISD11 amino acid residues critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these critical ISD11 residues to alanine leads to its compromised interaction with NFS1, which results in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Moreover, our findings highlight that, COXPD19 associated R68L ISD11 mutant displays reduced affinity to form a stable sub-complex with NFS1, thereby fails to prevent NFS1 aggregation, resulting impairment of Fe-S cluster biogenesis. The prime affected machinery is the ETC complex which demonstrates compromised redox properties, causing diminished mitochondrial respiration in COXPD19 patients. In summary, our findings provide compelling evidence that respiration defect due to impaired biogenesis of Fe-S clusters in ISCU myopathy patients, leads to manifestation of complex clinical symptoms. Additionally, our study highlights the role of ISD11 protein in Fe-S cluster biogenesis and maps the surface residues of ISD11 protein that are involved in interaction with sulfur donor protein NFS1. Moreover, we have demonstrated the molecular basis of disease progression of COXPD19 as a result of R68L ISD11 mutation.

In 1996, scientists discovered a connection between the gene for the human protein frataxin (FXN) and the neurodegenerative disease Friedreich’s ataxia (FRDA). Decreased FXN levels result in a variety of aberrant phenotypes including loss of activity for iron-sulfur containing enzymes, mitochondrial iron accumulation, and susceptibility to oxidative stress. These symptoms are the primary focus of current therapeutic efforts. In contrast our group is pursuing an alternate strategy of first defining FXN function at a molecular level then using this information to identify small molecule functional replacements. Recently, our group has discovered that FXN functions as an allosteric activator for the human Fe-S cluster assembly complex. The work presented here helps to further define molecular details of FXN activation and explain how FRDA missense mutants are functionally compromised. First, the FRDA missense mutants L182H and L182F were investigated. Unlike other characterized FRDA missense mutants, the L182F variant was not compromised in its ability to bind and activate the Fe-S assembly complex. The L182H variant exhibited an altered circular dichroism signature; suggesting a change in secondary structure relative to native FXN, and rapidly degraded. Together these studies suggest that L182 variants are less stable than native FXN and are likely prone to degradation in FRDA patients. Second, as a regulatory role of FXN suggests that its function is likely controlled by environmental stimuli, different maturation forms of FXN as well as post-translational modification mimics were tested as mechanisms to control FXN regulation. Here experiments were designed to test if a larger polypeptide form of FXN represents a functional form of the protein. Kinetic and analytical ultracentrifugation studies revealed a complex heterogeneous mixture of species some of which can activate the Fe-S assembly complex. A previously identified acetylation site was also tested using mutants that mimic acetylation. These mutants had little effect on the ability of FXN to bind and activate the assembly complex. Third, mutagenesis experiments were designed in which the FXN surface residues were replaced with alanine and the resulting variants were tested in binding and activity assays. These experiments revealed a localized “hot-spot” on the surface of FXN that suggests small cyclic peptide mimics might be able to replace FXN and function as FRDA therapeutics. Unexpectedly, one of the FXN variants exhibited significantly tighter binding and could have relevance for therapeutic development.

Human cytosolic monothiolglutaredoxin-3 (GLRX3) is a protein essential for the maturation of cytosolic [4Fe−4S] proteins. We show here that dimeric cluster-bridged GLRX3 transfers its [2Fe−2S]2+ clusters to the human P-loop NTPase NUBP1, an essential early component of the cytosolic iron−sulfur assembly (CIA) machinery. Specifically, we observed that [2Fe−2S]2+ clusters are transferred from GLRX3 to monomeric apo NUBP1 and reductively coupled to form [4Fe−4S]2+ clusters on both N-terminal CX13CX2CX5C and C-terminal CPXC motifs of NUBP1 in the presence of glutathione that acts as a reductant. In this process, cluster binding to the C-terminal motif of NUBP1 promotes protein dimerization, while cluster binding to the N-terminal motif does not affect the quaternary structure of NUBP1. The cluster transfer/assembly process is not complete on both N- and C-terminal motifs and indeed requires a reductant stronger than GSH to increase its efficiency. We also showed that the [4Fe−4S]2+ cluster formed at the N-terminal motif of NUBP1 is tightly bound, while the [4Fe−4S]2+ cluster bound at the C-terminal motif is labile. Our findings provide the first evidence for GLRX3 acting as a [2Fe−2S] cluster chaperone in the early stage of the CIA machinery. Iron-sulfur (Fe-S) clusters are among the most versatile cofactors in biology. Although Fe-S clusters formation can be achieved spontaneously in vitro with inorganic iron and sulfur sources, the in vivo behaviour is more complex and requires the so-called Fe-S biogenesis machineries. In the cytosol, the biogenesis of Fe-S proteins is assisted by the cytosolic Fe-S protein assembly machinery, which comprises at least thirteen known proteins, among which there are human ORAOV1 and YAE1. A hetero-complex formed by the two latter proteins facilitates Fe-S cluster insertion in the human ABC protein ABCE1 within a chain of binding events that are still not well understood. In the present work, ORAOV1 and the YAE1-ORAOV1 complex were produced and their structural and cluster binding properties spectroscopically investigated. It resulted that both ORAOV1 and the YAE1-ORAOV1 complex are characterized by well-structured alpha-helical regions and by unstructured, flexible regions, and are both able to bind a [4Fe-4S]2+ cluster. Bioinformatics and site-directed mutagenesis studies indicated that ORAOV1, and not YAE1, is the protein involved in [4Fe-4S]2+ cluster binding in the hetero-complex. ORAOV1 has indeed a conserved cluster-binding motif able to coordinate a [4Fe-4S] cluster. Overall, our data suggested that the YAE1-ORAOV1 complex might actively participate in the Fe-S cluster insertion into ABCE1 thanks to the [4Fe-4S]2+ cluster binding properties of ORAOV1.

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.