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Electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for obtaining structural information about chemical centres with unpaired electrons. In complex biological systems, EPR spectroscopy can be used to probe these paramagnetic centres and the long-range interactions between them. This thesis investigates two important types of enzymes, and in particular the role of the iron-sulfur electron-transfer centres they contain, with a variety of EPR techniques. Complex I (NADH:Ubiquinone Oxidoreductase) plays a key role in the electron transfer chain essential to the formation of ATP, and its malfunction has been related to numerous human diseases. It is a giant enzyme that contains the longest relay of iron-sulfur clusters known. EPR experiments conducted on complex I from bovine mitochondria yield crucial insight into the mechanism of efficient long-range electron transfer and bring us a step closer to understanding the functioning of this important complex. Hydrogenases are produced by micro-organisms and catalyse the reversible oxidation of H2. Most hydrogenases, including Hyd-2 from Escherichia coli, are very air-sensitive, but some, including E. coli Hyd-1 and Salmonella Hyd-5, are able to function in the presence of atmospheric levels of O2. Understanding the origins of this 'O2-tolerance' is of paramount importance if hydrogenases are to be exploited in future energy technologies. In this thesis, native E. coli Hyd-1 and Hyd-2, Salmonella Hyd-5, as well as O2-tolerant and O2-sensitive variants of E. coli Hyd-1 are characterised using EPR. The EPR investigations elucidate properties of the active site and the electron-transfer relay and, in conjunction with other techniques, reveal structural and mechanistic details of how a highly unusual iron-sulfur cluster in the electron-transfer chain enables some hydrogenases to sustain catalytic activity in the presence of O2.

DNA is constantly damaged by a variety of exogenous and endogenous sources. To maintain the integrity of the genome, different DNA repair mechanisms have evolved, which deal with different kinds of DNA damage. One of the DNA repair pathways, Nucleotide Excision Repair (NER), is highly conserved throughout the three kingdoms of life and deals mainly with lesions arising in the DNA duplex after exposure to UV-light. The NER pathway in archaea is more closely related to that of eukarya, although the overall process is not yet well understood. This thesis describes the isolation and characterisation of one of the repair factors, XPD, from the crenarchaeon Sulfolobus acidocaldarius (SacXPD). SacXPD was first identified due to its homology with the eukaryal XPD protein. In eukarya XPD is the 5a' -> 3a' helicase involved in opening the DNA duplex around a damaged site. In eukarya, XPD is part of a 10-subunit complex, where it fulfils important structural roles and takes part in NER, transcription initiation from RNA polymerase II promoters and cell cycle regulation. The archaeal protein on the contrary is a monomer and a 5a' -> 3a' SF2 DNA helicase as its eukaryal counterpart. Its cellular functions, however, are unclear. Upon purification of SacXPD, it was discovered that the protein binds an ironsulphur cluster (FeS), which is essential for its helicase activity, but not for any other enzymatic functions, such as the ATP hydrolysing activity. The FeS cluster domain was not only identified in archaeal XPD, but also in eukaryal XPD and other related eukaryal helicases, such as FancJ. The presence of the FeS cluster was confirmed in the eukaryotic XPD homologue Rad3 from Saccharomyces cerevisiae. Mutagenesis studies were used to investigate a possible function of the FeS cluster, which may be used to engage ssDNA during the duplex unwinding process. This would actively distort the ss/ ds DNA junction. In addition, the resulting bending of the clamped single DNA strand could be used to avoid reannealing. The consequences of some human mutations introduced into the SacXPD gene were investigated and could contribute to our understanding of the development of human diseases.

Trichomonas vaginalis is flagelated microaerophilic protozoan parasite from Excavata group, which causes trichomoniasis, the most common nonviral sexually transmitted disease in the world. It causes vaginitis in women and uretritis in man and it can also cause problems for example during pregnancy. This thesis is aimed on the characterisation of hydrogenosomal iron-sulfur flavoproteins (ISF) from Trichomonas vaginalis, proteins, which were only recently discovered in the proteome of hydrogenosome of T. vaginalis. Specifically, we have focused on characterisation of ISF3 which is, according to our data, active homodimer and binds flavin mononucleotide (FMN) and iron-sulphur centre in its active site. The iron- sulphur centre is not characterised yet. ISF3 is able to reduce oxygen, hydrogen peroxide, sodium nitrate and metronidazole also in the enzymatic system with PFO and ferredoxin. Next, I tried to reduce ammonium sulphate with ISF3, but unsuccessfully. These results correspond with the activities obtained for ISF from Methanosarcina thermophila, where ISF reduces oxygen and hydrogen peroxide to water. In addition, ISF3 is able to reduce nitrogen compounds. It is important according to the fact, that metronidazole is a drug from the group of 5−nitroimidazoles. The other results show the decrease...

CO2 fixation is a thermodynamically and kinetically challenging process, but nature has its own way of transforming CO2 into diverse organic molecules. Of our particular interest is 2-oxoacid:ferredoxin oxidoreductase (OFOR) that catalyzes the anaerobic, reversible inter-conversion of 2-oxoacids and CO2, making use of a small electron-transfer protein, ferredoxin (Fd), as the redox partner. This dissertation characterizes OFORs and Fds from organisms that exhibit different metabolic patterns and investigates how the interplay of OFOR and Fd could impact the fate of CO2 metabolism, asking the question What controls the catalytic bias of OFOR for CO2 evolution versus fixation? The study of OFORs and Fds from Desulfovibrio africanus and Hydrogenobacter thermophilus through an electrocatalytic assay reveals that the reduction potential of Fd is possibly associated with the biological function of OFOR and that CO2 fixation requires a low-potential electron donor. The Fd from H. thermophilus (HtFd1) is used as a model to probe the factors that govern iron-sulfur cluster potential. The dependence of OFOR activity on Fd potential is systematically studied with HtFd1 and its molecular variants through the electrocatalytic assay and a coupled enzyme assay. The results suggest there is a Fd “potential optimum” for OFOR-catalyzed CO2 fixation. The study of a 2-oxoglutarate:ferredoxin oxidoreductase (OGOR) and three Fds from Magnetococcus marinus MC-1 further highlights other factors such as the intramolecular electron-transfer within Fd and the electrostatic and hydrophobic interactions at the protein-protein interface in determining OFOR-Fd interaction. The characterization of an OGOR from M. marinus MC-1 (MmOGOR) also provides kinetic, structural and spectroscopic details for a CO2-fixing OFOR that contains only one iron-sulfur cluster. Overall, this work furthers the scientific understanding of how nature achieves CO2 fixation through supplying reducing equivalents and with enzymes as efficient catalysts, and how intermolecular electron-transfer mediated by protein-protein interaction could regulate enzyme catalysis. / 2019-10-08T00:00:00Z

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Le Nicotinamide Adénine Dinucléotide (NAD) est un cofacteur clé du métabolisme cellulaire. Synthétisé à partir d'acide quinolinique (QA) chez tous les organismes vivants, la biosynthèse du QA diffère entre les eucaryotes et les procaryotes. Chez les eucaryotes, il est produit à partir de L-tryptophane alors que chez les procaryotes et les plantes, il est synthétisé par l'action concertée de deux enzymes: la L-aspartate oxydase (NadB) qui permet la formation d'iminoaspartate (IA) à partir de L-aspartate et la quinolinate synthase (NadA) qui permet la condensation de deux molécules, la dihydroxyacétone-phosphate (DHAP) et l'iminoaspartate, pour former l'acide quinolinique. En plus de cette voie dite « de novo », la plupart des organismes possèdent une voie de secours qui produit le NAD à partir de niacine provenant de l'alimentation ou de la dégradation du NAD. Chez certains pathogènes tels que Mycobacterium leprae et Helicobacter pylori, cette voie de secours n'existe pas. Ceci fait de NadA une cible particulièrement attractive pour la conception d'antibactériens et ceci d'autant plus qu'elle est absente chez l'homme.NadA est la seule enzyme de la voie de biosynthèse de novo du NAD dont le mécanisme moléculaire et la structure tridimensionnelle sous forme active (avec son centre [4Fe-4S]2+) sont inconnus. Grâce à l'utilisation d'analogues de substrats ou d'intermédiaires réactionnels, nous avons pu non seulement avancer dans l'élucidation du mécanisme moléculaire de NadA et notamment dans la compréhension du rôle du centre [4Fe-4S]2+ dans la catalyse mais en plus, nous avons été en mesure de proposer un 1er inhibiteur in vitro et in vivo de NadA : l'acide 4,5 Dithiohydroxyphtalique (DTHPA). Le DTHPA nous a fourni de bonnes bases pour la conception d'inhibiteurs puissants et spécifiques de NadA grâce à une étude Structure-Activité. Par ailleurs, nous avons résolu la 1ère structure aux rayons X de NadA sous forme holoprotéine dont les données structurales nous ont grandement aidé dans la compréhension du mécanisme de NadA. / The Nicotinamide Adenine Dinucleotide (NAD) is a key cofactor essential for cellular metabolism. Synthesized from quinolinic acid (QA) in all living organisms, NAD biosynthesis is different between eucaryotes and procaryotes. Indeed, most of eukaryotes produce QA from L-tryptophan, whereas most of prokaryotes and plants synthesize QA by the concerted action of 2 enzymes: L-aspartate oxydase (NadB), an FAD enzyme, which catalyzes L-Aspartate oxidation to form iminoaspartate (IA) while quinolinate synthetase (NadA) allows condensation between IA and Dihydroxyacetone Phosphate (DHAP) to produce QA. Besides this « de novo » pathway, most eukaryotes and some bacteriae have a salvage pathway which allows NAD synthesis from nutrients and metabolites of NAD degradation in order to maintain a correct pool of NAD in the cell. However, some pathogens like Mycobacterium leprae, Helicobacter pylori do not possess this pathway. As a consequence, NadA represents a very attractive target for designing specific antibacterial agents since it does not exist in Human.NadA is the only metalloenzyme of NAD de novo biosynthesis whose molecular mechanism and tridimensional structure with its [4Fe-4S]2+ cluster are unknown. Using substrate and intermediate analogues, we have been able to understand better NadA mechanism, especially [4Fe-4S]2+ cluster role in catalysis. Moreover, we proposed the first in vitro and in vivo inhibitor of NadA : the 4,5 Dithiohydroxyphtalic Acid (DTHPA) which gave us basis to design powerful and specific NadA inhibitors thanks to a structure-activity relationship study. Besides, we resolved the first X-rays structure of NadA under its holoprotein form. Datas we extracted from it helped us greatly to understand NadA mechanism.

In der vorliegenden Arbeit wurde der ATP-gekoppelte uphill Elektronentransfer von reduziertem RACo auf Kobalt(II)-CoFeSP untersucht. Dazu wurden zunächst die Bedingungen der rekombinanten Genexpression in Escherichia coli und die Reinigungsstrategie der Proteine verbessert, um einen Cofaktorgehalt beider Proteine von annähernd 100 % zu erreichen. Anschließend wurden die Reaktionsbedingungen des Elektronentransfers optimiert, um eine tiefergehende Analyse zu ermöglichen. Die Ergebnisse dieser Arbeit deuten darauf hin, dass durch die Bindung von ATP ein bidirektionaler Elektronentransfer induziert wird. Der Elektronentransfer konnte mit nicht-hydrolysierbaren ATP-Analoga und mit ADP induziert werden. Weder für die nicht-hydrolysierbaren ATP-Analoga noch für ADP konnten anschließend Hydrolyseprodukte nachgewiesen werden. Zusätzlich konnte für die limitierende Rate der ATP-Hydrolyse ein mehr als 100-fach kleinerer Wert bestimmt werden als für den Elektronentransfer. Beide Ergebnisse zeigen, dass der Elektronentransfer unabhängig von der ATP-Hydrolyse ist. Kobalt(I)-CoFeSP kann jedoch auch ein Elektron auf oxidiertes RACo übertragen, was auf einen bidirektionalen Elektronentransfer hindeutet. Diese These wurde mit der Beobachtung untermauert, dass sich durch Zugabe von ADP und der Erhöhung der ADP-Konzentration die Anzahl der transferierten Elektronen pro CoFeSP zunimmt und sich somit die Lage des entstehenden Gleichgewichts verschieben lässt. Auf dieser Datengrundlage konnten drei mögliche Modelle für den Reaktionsmechanismus erstellt werden, von welchen ein Modell als am wahrscheinlichsten erscheint. In diesem Reaktionsmechanismus gleichen sich die Redox-Potentiale beider Redox-Zentren durch die ATP-Bindung an. Dies ermöglicht den Elektronentransfer vom [2Fe2S]-Cluster von RACo auf das Kobalt-Ion des Cobalamins. Die Rückreaktion wird durch eine erneute Reduktion des [2Fe2S]-Clusters verhindert und durch die anschließende ATP-Hydrolyse dissoziiert der Komplex. / In the present work, ATP-coupled uphill electron transfer from reduced RACo to cobalt(II)-CoFeSP was investigated. For this purpose, the conditions of recombinant gene expression in Escherichia coli and the purification strategy of the proteins were improved to achieve a cofactor content of both proteins close to 100%. Subsequently, the electron transfer reaction conditions were optimized to enable a more in-depth analysis. The results of this work indicate that a bidirectional electron transfer is induced by the binding of ATP. Electron transfer could be induced with non-hydrolysable ATP analogues and with ADP. Neither for the nonhydrolyzable ATP analogues nor for ADP hydrolysis products could subsequently be detected. In addition, a value more than 100-fold smaller could be determined for the limiting rate of ATP hydrolysis than for electron transfer. Both results indicate that electron transfer is independent of ATP hydrolysis. However, cobalt(I)-CoFeSP can also transfer an electron to oxidized RACo, suggesting bidirectional electron transfer. This hypothesis was supported with the observation that adding ADP and increasing the ADP concentration increases the number of transferred electrons per CoFeSP by shifting the position of the emerging equilibrium. Based on these data, three possible models for the reaction mechanism are suggested, of which one model appears to be the most plausible. In this reaction mechanism, the redox potentials of both redox centers equalize due to ATP binding. This allows electron transfer from the [2Fe2S] cluster of RACo to the cobalt ion of cobalamin. The back reaction is prevented by a further reduction of the [2Fe2S] cluster, and subsequent ATP hydrolysis dissociates the complex

Metabolism is a subset of chemistry that allows cells to defy thermodynamic equilibrium, a fundamental process that must have been in place from the very beginning of biology. Before evolution produced efficient catalysts in the form of complex protein machinery, short metal binding peptides might have preceded modern metalloproteins. Such prebiotic, metal-binding motifs have been hypothesized to have existed through analyses of extant protein sequences. However, it is unclear how metal-binding motifs might have evolved in the harsh prebiotic environment. Here, we show how certain environments, in particular seawater-like environments rich in divalent cations and especially Mg2+, support the survival of short peptides upon extreme temperatures as high as 150 °C. Moreover, while Mg2+ does not offer the same protection from UV light, peptides are protected from both heat and irradiation when bound to a metal ion. The results suggest that specific environments rich in metal ions may be better suited for the emergence of complex systems in the path toward life. Additionally, the conditional degradation of peptides depending on their ability of binding metals might have enabled a selection mechanism that would favor the survival of metal-binding motifs which resemble the motifs found in modern proteins. These short sequences could have acted as early, simple catalysts able to facilitate a restricted set of chemical reactions, which would shape the emergence and biology of the Last Universal Common Ancestor.