Coupled electron and proton transfer (CEPT) events are fundamental for many bioenergetic conversions that involve redox reactions. Understanding the details underlying CEPT processes will advance our knowledge of (1) how nature regulates energy conversion; (2) our strategies for achieving renewable energy sources; (3) how to cope with CEPT dysfunction diseases. Studies of the detailed mechanism(s) of CEPT in biological systems is challenging due to their complex nature. Consequently, controversies between the concerted and sequential mechanism of CEPT for many systems remain. This dissertation focuses on the bovine mitochondrial cytochrome bc1 complex. CEPT in the bc1 complex operates by a modified "Q-cycle"(1) and catalyzes electron transfer from ubiquinol (QH2), to cyt c via an iron sulfur cluster (ISC) and to the low potential hemes of cyt b, where it reduces ubiquinone (UQ). The electron transfer is coupled to the translocation of protons across the mitochondrial inner membrane, generating a proton gradient that drives ATP synthesis. Although the Q-cycle is widely accepted as the model that best describes how electrons and protons flow in bc1, detailed binding geometries at the Qo site (QH2 oxidation site) and Qi site (UQ reduction site) remain controversial. The binding geometries play critical roles in the thermodynamic and/or kinetic control of the reaction and protonatable amino acid side chains can participate in the proton transfer. The central focuses of this dissertation are molecular dynamics simulations of cofactor binding geometries near the Qo and Qi sites, calculations of the pKa values of ionizable amino acid side chains implicated in cofactor binding, especially the ISC-coordinated histidines, and implications for the proposed mechanism(s) of CEPT. For the first time, pKa values of the ISC-coordinated histidines are differentiated. The computed pKa values of 7.8±0.5 for His141 and 9.1±0.6 for His161 agree well with experiment (7.63±0.15 and 9.16±0.28). Thus, His161 should be protonated at physiological pH and cannot be the first proton acceptor in the QH2 oxidation. Water mediated hydrogen bonds between substrate models and the protein and water accessibility to the Qo and Qi sites were maintained in simulations, implying that water molecules are likely the proton donors and acceptors. / Bayer School of Natural and Environmental Sciences; / Chemistry and Biochemistry / PhD; / Dissertation;

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.

The cytochrome bc1 complex of the mitochondrial electron transport chain accomplishes the enzymatic reaction known as the modified Q-cycle. In the Q-cycle the bc1 complex transports protons from the matrix to the intermembrane space of the mitochondria, creating the proton gradient used to make ATP. The energy to move these protons is obtained by shuttling electrons from the coenzyme ubiquinol (QH2) to coenzyme ubiquinone (Q) and the mobile cytochrome c. This well studied complex is ideal for examining molecular adaptation because it consists of ten different subunits, it functions as a dimer, and it includes at least five different active sites. The program TreeSAAP was used to characterize molecular adaptation in the bc1 complex and identify specific amino acid sites that experienced positive destabilizing (radical) selection. Using this information and three-dimensional structures of the protein complex, selection was characterized in terms of coevolution and coadaptation. Coevolution is described as reciprocal local biochemical shifts based on phylogenetic location and results in overall maintenance. Coadaptation, on the other hand, is more dynamic and is described as coordinated local biochemical shifts based on phylogenetic location which results in overall adaptation. In this study both coevolution and coadaptation were identified in various locations on the protein complex near the active sites. Sites in the pore region of cyt c1 were shown to exhibit coevolution, in other words maintenance, of many biochemical properties, whereas sites on helix H of cyt b, which flanks the active sites Qo and Qi, were shown to exhibit coadaptation, in other words coordinated shifts in the specific properties equilibrium constant and solvent accessible reduction ratio. Also, different domains of the protein exhibited significant shifts in drastically different amino acid properties: the protein imbedded in the membrane demonstrated shifts in mainly functional properties, while the part of the complex in the intermembrane space demonstrated shifts in conformational, structural, and energetic properties.

Zahlreiche strikt anaerob lebende Mikroorganismen, darunter acetogene Bakterien, Sulfatreduzierer und methanogene Archaeen, nutzen den reduktiven Acetyl-CoA-Weg zur autotrophen Kohlenstoff-Fixierung oder Energiegewinnung. Die letzten Schritte der Acetyl-CoA-Bildung beruhen hierbei auf dem Zusammenspiel dreier Proteine, dem Corrinoid-Eisen/Schwefel-Protein (CoFeSP), der Methyltetrahydrofolat:CoFeSP-Methyltransferase (MeTr) und dem Acetyl-CoA-Synthase/CO-Dehydrogenase-Komplex (ACS/CODH). In der vorliegenden Arbeit wurde die Substratbindung an MeTr durch thermodynamische und kinetische Messungen untersucht. MeTHF bindet stärker an das Enzym als das demethylierte Produkt Tetrahydrofolat (THF) und scheint dabei einem einstufigen Bindungsmodell zu folgen. Das Substrat wird bei der Bindung an MeTr protoniert, wobei Asn200 eine protonierte H-N5(+)-CH3-Position des MeTHF durch eine alternative Konformation stabilisieren könnte. Asp44 und Asp76 bilden eine funktionelle Dyade bei der Substratbindung, kommen als Protondonoren zur Substrataktivierung jedoch nicht in Frage. Die Kristallstruktur von CoFeSP wurde erstmals vollständig mit der flexiblen N-terminalen [4Fe4S]-Cluster-Bindedomäne bestimmt. Die für die Cobalamin-Bindedomäne erwarteten Konformationsänderungen wurden anhand der Interaktion mit dem reduktiven Aktivator von CoFeSP (RACo) analysiert. Durch Förster-Resonanzenergietransfer wurde eine Annäherung der ortsspezifisch markierten CoFeSP-Positionen beobachtet und anhand des Fluoreszenzsignals die Kinetik der Komplexbildung mit RACo bestimmt. Durch gepulste Elektronendoppelresonanz konnte ebenfalls eine Abstandsänderung nachgewiesen werden. ACS wurde als apo-Enzym gereinigt und durch NiCl2-Rekonstitution in die aktive Form überführt. Durch die Kristallisation der C-terminalen ACS-Domäne wurden hochaufgelöste Strukturen erzeugt, welche eine Diskussion der strukturellen Details des aktiven Zentrums ermöglichen. / Several anaerobic microorganisms, including acetogenic bacteria, sulfate-reducing bacteria and methanogenic archaea operate the reductive acetyl-CoA pathway for autotrophic carbon fixation or to gain energy. The last steps of acetyl-CoA formation rely on three enzymes, the corrinoid-iron/sulfur-protein (CoFeSP), the methyltetrahydrofolate:CoFeSP methyltransferase (MeTr) and the acetyl-CoA synthase/CO dehydrogenase complex (ACS/CODH). Substrate binding to MeTr was investigated by thermodynamic and kinetic meassurements. MeTHF binds slightly stronger than the demethylated product tetrahydrofolate (THF), likely following a simple one-step-binding mechanism. Substrate binding to MeTr is coupled to proton uptake. A H-N5(+)-CH3-transition state of MeTHF could be stabilized by an alternative conformation of Asn200. Asp44 and Asp76 form a functional dyade in substrate binding but can be excluded as proton donors for substrate activation. The crystal structure of CoFeSP was solved completely, including the previously disordered N-terminal [4Fe4S]-cluster binding domain. The expected conformational change of the corrinoid binding domain was characterized by analyzing the interaction between CoFeSP and its reductive activator (RACo). An approach of the labeled CoFeSP positions in the CoFeSP:RACo complex was observed by Förster resonance energy transfer. Based on the corresponding fluorescence signal, the kinetics of complex formation were meassured in solution. Pulsed electron double resonance also showed that the labeled positions approach upon complex formation. Full-length ACS was purified in the apo state. A reconstitution of the A-cluster with NiCl2 resulted in active enzyme. Different crystal structures of the isolated C-terminal domain of ACS were solved at high resolution. Therefore, structural details of the active site could be discussed.

Die Verknüpfung einer exergonischen mit einer endergonischen Reaktion zur Ermöglichung der letzteren ist eine in biologischen Systemen weit verbreitete Strategie. Energetisch benachteiligte Elektronenübertragungsreaktionen im Rahmen der reduktiven Aktivierung von Nitrogenasen, Radikal-abhängigen β,α-Dehydratasen, der zu diesen verwandten Benzoyl-CoA-Reduktasen und diversen Cobalamin-abhängigen Methyltransferasen sind gekoppelt an die Hydrolyse von ATP. Der Methylgruppentransfer des reduktiven Acetyl-CoA-Weges von Carboxydothermus hydrogenoformans erfordert den Co(I)-Zustand des Corrinoid/Eisen-Schwefel Proteins (CoFeSP). Um diese superreduzierte Form nach einer oxidativen Inaktivierung zu regenerieren ist ein „Reparaturmechanismus“ erforderlich. Ein offenes Leseraster (orf7), welches möglicherweise für eine reduktive Aktivase von Corrinoid Enzymen (RACE) kodiert, wurde in dem Gencluster der am reduktiven Acetyl-CoA-Weg beteiligten Proteine entdeckt. Im Rahmen dieser Arbeit wurde dieses potenzielle RACE Protein biochemisch und strukturell charakterisiert und die ATP-abhängige reduktive Aktivierung von CoFeSP untersucht. Auf Grundlage der in dieser Arbeit gewonnenen Ergebnisse wurde ein Mechanismus für die ATP-abhängige Aktivierung entworfen. Dieser gibt Einblicke wie die durch ATP-Hydrolyse bereitgestellte Energie einen energetisch ungünstigen Elektronentransfer ermöglichen kann. Hierzu kombiniert RACo das Ausgleichen von Bindungsenergien mit Modulationen am Elektronenakzeptor. Eine vergleichbare Strategie wurde bisher in keinem anderen ATP-abhängigen Elektronenübertragungssystem wie dem von Nitrogenasen, Radikal-abhängigen β,α-Dehydratasen oder Benzoyl-CoA-Reduktasen beobachtet und könnte ein für RACE Proteine allgemein gültige Eigenschaft darstellen. / The principle of coupling an exergonic to an endergonic reaction to enable the latter is a widespread strategy in biological systems. Unfavoured electron transfer reactions in the reductive activation of nitrogenases, radical-dependent β,α-dehydratases and the related benzoyl- CoA reductases, as well as different cobalamin-dependent methyltransferases are coupled to the hydrolysis of ATP. The reductive acetyl-CoA pathway of Carboxydothermus hydrogenoformans relies on the superreduced Co(I)-state of the corrinoid/iron-sulfur protein (CoFeSP) that requires a “repair mechanism” in case of incidental oxidation. An open reading frame (orf7) coding for a putative reductive activase of corrinoid enzymes (RACE) was discovered in the gene cluster of proteins involved in the reductive acetyl-CoA pathway. In this work, this putative RACE protein was biochemically and structurally characterised and the ATP-dependent reductive activation of CoFeSP was investigated. Based on the results of this study, a mechanism for the ATP-dependent reactivation of CoFeSP was deduced providing insights into how the energy provided by ATP could trigger this unfavourable electron transfer. The reductive activator of CoFeSP combines balance of binding energies and modulations of the electron acceptor to promote the uphill electron transfer to CoFeSP. A comparable strategy has not been observed in other ATP-dependent electron transfer systems like nitrogenases, radical-dependent β,α-dehydratases and benzoyl- CoA reductases and could be a universal feature of RACE proteins.