Mitochondria contain their own genetic system, encoding key subunits of the oxidative phosphorylation system. These subunits are expressed by an organelle-specific gene expression machinery. This work revealed a number of fundamental aspects of mitochondrial gene expression and provides evidence that this process is organized in a unique and organelle-specific manner which likely evolved to optimize protein synthesis and assembly in mitochondria. Most importantly, improving the experimental handling of ribosomes we could show that mitochondrial ribosomes are organized in large assemblies that we termed MIOREX complexes. Ribosomes present in these complexes organize gene expression by recruiting multiple factors required for post-transcriptional steps. In addition, we could reveal mechanisms by which ribosome-interactor complexes modulate and coordinate the expression and assembly of the respiratory chain subunits. For example we showed that the Cbp3-Cbp6 complex binds to the ribosome in proximity to the tunnel exit to coordinate synthesis and assembly of cytochrome b. This location perfectly positions Cbp3-Cbp6 for direct binding to newly synthesized cytochrome b and permits Cbp3-Cbp6 to establish a feedback loop that allows modulation of cytochrome b synthesis in response to assembly efficiency. Likewise the interaction of the membrane-anchor proteins Mba1 and Mdm38 with the tunnel exit region enables them to participate in the translation of the two intron-encoding genes COX1 and COB in addition to their role in membrane insertion.  In summary, work presented in this thesis shows that mitochondrial gene expression is a highly organized and regulated process. The concepts and technical innovations will facilitate the elucidation of many additional and important aspects and therefore contribute to the general understanding of how proteins are synthesized in mitochondria. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Des inhibiteurs du complexe bc1 de la chaîne respiratoire mitochondriale ont été développés comme agents antimicrobiens pour lutter contre des pathogènes de l’Homme et de plantes. Ces drogues ciblent les poches catalytiques Qo et Qi formées par le cytochrome b. La comparaison de séquences de cette protéine montre que les sites Qo et Qi sont bien conservés entre les organismes mais qu’il existe toutefois des variations qui pourraient expliquer leur différence de sensibilité aux drogues. A l’aide du modèle levure S. cerevisiae, nous avons étudié les déterminants de la résistance/sensibilité naturelle à deux antipaludiques se liant au site Qo de Plasmodium: l’atovaquone et RCQ06. Nous avons notamment montré que le résidu 275 joue un rôle clé dans ce phénomène. Une approche similaire est actuellement utilisée pour identifier les facteurs de la sensibilité différentielle à deux drogues ciblant le site Qi des oomycètes. Malheureusement, des cas de résistance acquise à ces antimicrobiens ont été rapportés et ont pour origine des mutations dans le cytochrome b. De ce fait, de nouvelles molécules sont requises pour court-circuiter ces résistances. Au cours de ma thèse, nous avons mis au point un test qui permet de cribler des molécules capables d’inhiber la fonction respiratoire. Nous avons ainsi pu identifier un nouvel inhibiteur du complexe bc1 : D12. Nous avons ensuite déterminé le mode de liaison de cette molécule ainsi que celui d’un composé capable d’inhiber la prolifération de Plasmodium, HDQ, grâce à une collection de mutants des poches catalytiques. HDQ s’est avéré être un inhibiteur du site Qi. Il pourrait être utilisé avec un inhibiteur du site Qo afin de limiter l’apparition de mutations de résistance. D12 est un inhibiteur du site Qo qui est capable notamment de court-circuiter la mutation de résistance à des fongicides du site Qo G143A. Cette dernière a été trouvée chez de nombreux phytopathogènes, mais n’est cependant pas apparue chez des champignons possédant un intron immédiatement après le codon codant pour la glycine 143. En utilisant la levure, nous avons montré que la mutation empêche l’épissage de l’intron en altérant la structure exon/intron. Nous avons également identifié des mécanismes de « by-pass » qui permettent de restaurer la fonction respiratoire du mutant et qui pourraient apparaître chez les pathogènes. Les mutants créés au cours de ma thèse pourront aider à identifier, concevoir et caractériser de nouveaux antimicrobiens et à étudier l’apparition de mutations de résistance. / Inhibitors of the mitochondrial respiratory chain bc1 complex are currently used against human and plant pathogens. These drugs bind to Qo and Qi pockets of the mitochondrially-encoded cytochrome b. Comparison of the cytochrome b sequences shows that the Qo and Qi sites are well conserved between organisms. However, there are variations that could explain the differential sensitivity to respiratory inhibitors. In order to investigate the determinants of resistance / sensitivity to the antimalarial compounds, atovaquone and RCQO6, we used S.cerevisiae as a model. We showed that residue 275 plays a central role in the sensitivity to these drugs. We are now using a similar approach to identify the determinants of sensitivity towards two drugs targeting the oomycete Qi site. Unfortunately, cases of acquired resistance to these antimicrobial agents have been reported. They are caused by mutations in the cytochrome b. Thus, new molecules are required to bypass resistance. During my PhD, we developed a test to screen chemical libraries and identify inhibitors of the respiratory function. We identified a novel inhibitor of bc1 complex: D12. We determined the binding mode of D12 as well as of HDQ, a compound capable of inhibiting the proliferation of Plasmodium. To do this, we used a collection of mutants with alterations of the catalytic pockets. We showed that HDQ targets the Qi site. This finding suggests that HDQ could be used with an inhibitor of the Qo site to limit the emergence of resistance mutations. D12 is an inhibitor of Qo site and fully active against the enzyme harbouring the fungicide resistance mutation G143A. This mutation has been reported in many plant pathogenic fungi but has not evolved in fungi that harbour an intron immediately after the codon for G143. Using yeast, we showed that the mutation hinders the splicing of this intron by altering the exon / intron structure needed for efficient intron excision. We also identified by-pass mechanisms that restore respiratory function of the G143A mutant. These mechanisms identified in yeast could potentially arise in pathogenic fungi. Mutants created during my PhD will help to identify, design and characterize new drugs and to study the emergence of resistance mutations.

The inner membrane of mitochondria harbors the complexes of the respiratory chain and the ATP synthase, which perform the key metabolic process oxidative phosphorylation. These complexes are composed of subunits from two different genetic origins: the majority of constituents is synthesized on cytosolic ribosomes and imported into mitochondria, but a handful of proteins, which represent core catalytic subunits, are encoded in the organellar DNA and translated on mitochondrial ribosomes. Using yeast as a model organism, I investigated the mitochondrial ribosomal tunnel exit, the region of the ribosome where the nascent chain emerges and that in cytosolic ribosomes serves as a platform to bind biogenesis factors that help the newly synthesized protein to mature. This study provided insights into the structural composition of this important site of mitochondrial ribosomes and revealed the positioning of Cbp3 at the tunnel exit region, a chaperone required specifically for the assembly of the bc1 complex. In my further work I found that Cbp3 structurally and functionally forms a tight complex with Cbp6 and that this complex exhibits fundamental roles in the biogenesis of cytochrome b, the mitochondrially encoded subunit of the bc1 complex. Bound to the ribosome, Cbp3-Cbp6 stimulates translation of the cytochrome b mRNA (COB mRNA). Cbp3-Cbp6 then binds the fully synthesized cytochrome b, thereby stabilizing and guiding it further through bc1 complex assembly. The next steps involve the recruitment of the assembly factor Cbp4 to the Cbp3-Cbp6/cytochrome b complex and presumably acquisition of two redox active heme b cofactors. During further assembly Cbp3-Cbp6 is released from cytochrome b, can again bind to the ribosome and activate further rounds of COB mRNA translation. The dual role of Cbp3-Cbp6 in both translation and assembly allows the complex to act as a regulatory switch to modulate the level of cytochrome b synthesis in response to the bc1 complex assembly process. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

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;

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.