Assembly of cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, requires a concerted activity of a number of chaperones and factors for the correct insertion of subunits, accessory proteins, cofactors and prosthetic groups. Most of the fundamental biological knowledge concerning mitochondrial copper homeostasis and insertion of copper into COX derives from investigations in the yeast Saccharomyces cerevisiae. In this organism, Cox17 was the first identified factor involved in this pathway. It is a low molecular weight protein containing highly conserved twin Cx9C motifs and is localized in the cytoplasm as well as in the mitochondrial intermembrane space. It was shown that copper-binding is essential for its function. So far, the role of Cox17 in the mammalian mitochondrial copper metabolism has not been well elucidated. Homozygous disruption of the mouse COX17 gene leads to COX deficiency followed by embryonic death, which implies an indispensable role for Cox17 in cell survival. In this thesis, the role of COX17 in the biogenesis of the respiratory chain in HeLa cells was explored by use of siRNA. The knockdown of COX17 results in a reduced steady-state concentration of the copper-bearing subunits of COX and affects growth of HeLa cells accompagnied by an accumulation of ROS and apoptotic cells. Furthermore, in accordance with its predicted function as a copper chaperone and its role in formation of the binuclear copper center of COX, COX17 siRNA knockdown affects COX-activity and -assembly. It is now well accepted that the multienzyme complexes of the respiratory chain are organized in vivo as supramolecular functional structures, so called supercomplexes. While the abundance of COX dimers seems to be unaffected, blue native gel electrophoresis reveals the disappearance of COX-containing supercomplexes as an early response. Accumulation of a novel ~150 kDa complex containing Cox1, but not Cox2 could be observed. This observation may indicate that the absence of Cox17 interferes with copper delivery to Cox2, but not to Cox1. Data presented here suggest that supercomplex formation is not simply due to assembly of completely assembled complexes. Instead an interdependent assembly scenario for the formation of supercomplexes is proposed that requires the coordinated synthesis and association of individual complexes.:List of Figures and Tables Abbreviations Abstract 1 Indroduction 1.1 Mitochondria and the respriratory chain 1.2 The human mitochondrial genome 1.3 Homoplasmy and heteroplasmy 1.4 Mitochondrial disorders 1.4.1 Mutations in mitochondrial DNA 1.4.2 Mutations in nuclear DNA 1.5 Cytochrome c oxidase 1.6 Cytochrome c oxidase assembly 1.7 Copper and its trafficking in the cell 1.8 Mitochondrial copper metabolism 1.9 Cox17 1.10 Aims of the thesis 2 Materials and Methods 2.1 Materials 2.1.1 Chemicals and reagents 2.1.2 Antibodies 2.1.3 Plasmid 2.1.4 Kits 2.1.5 Marker 2.1.6 Enzymes 2.1.7 Primers 2.1.8 siRNAs 2.2 Methods 2.2.1 Cell culture 2.2.1.1 Cell culture: HeLa cells 2.2.1.2 Cell culture: HeLa cells transfected with pTurboRFP-mito 2.2.1.3 Subcultivation 2.2.1.4 Determination of cell number 2.2.1.5 Cell storage and thawing 2.2.2 Transient transfection of HeLa cells 2.2.3 Transfection of HeLa cells with pTurboRFP-mito 2.2.4 Immunocytochemistry 2.2.5 RNA extraction and quantitative real-time PCR 2.2.6 Isolation of mitochondria 2.2.6.1 Isolation of mitochondria for BN-PAGE Analysis 2.2.6.2 Isolation of mitochondria for localization studies 2.2.6.3 Isolation of bovine heart mitochondria 2.2.7 Proteinase K treatment of mitochondria and mitoplasts 2.2.8 Photometric activity assay 2.2.8.1 Citrate synthase activity 2.2.8.2 Cytochrome c oxidase activity 2.2.9 Blue native polyacrylamide gel electrophoresis (BN-PAGE) 2.2.9.1 In gel activity assay 2.2.9.2 2D-BN/SDS-PAGE 2.2.10 SDS-PAGE and Western blot analysis 2.2.11 Direct stochastic optical reconstruction microscopy (dSTORM) 2.2.12 Flow cytometric phenotyping 2.2.12.1 Determination of cell cyle phase 2.2.12.2 Identification of apoptotic cells 2.2.12.3 Detection of ROS 2.2.13 Oxygen measurement 2.2.14 Cu–His supplementation 3 Results 3.1 Subcellular localization of Cox17 3.2 Transient knockdown of COX17 in HeLa cells 3.2.1 Knockdown of COX17 mRNA 3.2.2 Knockdown of Cox17 protein 3.2.3 Effect of COX17 knockdown on the steady-state levels of OXPHOS subunits 3.2.4 Effect of COX17 knockdown on the steady-state levels of copperbearing COX subunits 3.2.5 Subdiffraction-resolution fluorescence imaging 3.3 Phenotypical characterization 3.3.1 Growth analyis 3.3.2 Cell cycle analysis 3.3.3 Apoptosis assay 3.3.4 Detection of ROS 3.3.5 Oxygen measurement 3.4 Cytochrome c oxidase activity 3.5 Characterization of mt OXPHOS complexes 3.5.1 BN-PAGE/in gel activity assays 3.5.2 Supramolecular organization of COX 3.5.3 Molecular organization of Cox17 3.5.4 Molecular organisation of copper-bearing COX subunits Cox1 and Cox2 3.5.5 Supramolecular organization of RC complexes 3.5.6 dSTORM of supercomplexes 3.6 Copper supplementation 4 Discussion 4.1 Dual localization of human Cox17 4.2 COX17 knockdown affects steady-state levels of copper-bearing COX subunits Cox1 and Cox2 4.3 Supramolecular organization of RC is affected as an early response to COX17 knockdown 4.4 Cox17 is primarily engaged in copper delivery to Sco1/Sco2 4.5 Copper supplementation alone cannot rescue the COX17 phenotype 4.6 Outlook 5 Appendix 6 PhD publication record 7 References

Mitochondria are cell organelles which play an essential role in the cell energy supply providing the universal high energetic molecule ATP which is used in numerous energy consuming processes. The core of the ATP production, oxidative phosphorylation (OXPHOS) consists of four enzyme complexes (respiratory chain) which establish, driven by redox reactions, a proton gradient over the inner mitochondrial membrane. The ATP-synthase uses this electrochemical gradient to phosphorylate ADP to ATP. Dysfunctioning of an OXPHOS complex can have severe consequences for the energy metabolism and cause rare but incurable dysfunctions in particular tissues with a high energy demand such as brain, heart, kidney and skeleton muscle. Moreover mitochondria are linked to widespread diseases like diabetes, cancer, Alzheimer and Parkinson. Further, reactive oxygen species which are a by-product of the respiratory chain, are supposed to play a crucial role in aging. The aim of this work is to provide a realistic model of OXPHOS which shall help understanding and predicting the interactions within the OXPHOS and how a local defect (enzyme deficiency or modification) is expressed globally in mitochondrial oxygen consumption and ATP synthesis. Therefore we chose a bottom-up approach. In a first step different types of rate equations were analyzed regarding their ability to describe the steady state kinetics of the isolated respiratory chain complexes in the absence of the proton gradient. Here Michaelis-Menten like rate equations were revealed to be appropriate for describing their behavior over a wide range of substrate and product concentrations. For the validation of the equations and the parameter estimation we have performed kinetic measurements on bovine heart submitochondrial particles. The next step consisted in the incorporation of the proton gradient into the rate equations, distributing its influence among the kinetic parameters such that reasonable rates were obtained in the range of physiological electrochemical potential differences. In the third step, these new individual kinetic rate expressions for the OXPHOS complexes were integrated in a global model of oxidative phosphorylation. The new model could fit interrelated data of oxygen consumption, the transmembrane potential and the redox state of electron carriers. Furthermore, flux inhibitor titration curves can be well reproduced, which validates its global responses to local effects. This model may be of great help to understand the increasingly recognized role of mitochondria in many cell processes and diseases as illustrated by some simulations proposed in this work. / Les mitochondries sont l’usine à énergie de la cellule. Elles synthétisent l’ATP à partir d’une succession de réactions d’oxydo-réduction catalysées par quatre complexes respiratoires qui forment la chaîne respiratoire. Avec la machinerie de synthèse d’ATP l’ensemble constitue les oxydations phosphorylantes (OXPHOS). Le but de ce travail est de bâtir un modèle des OXPHOS basé sur des équations de vitesse simples mais thermodynamiquement correctes, représentant l’activité des complexes de la chaîne respiratoire (équations de type Michaelis- Menten). Les paramètres cinétiques de ces équations sont identifiés en utilisant les cinétiques expérimentales de ces complexes respiratoires réalisées en absence de gradient de proton. La phase la plus délicate de ce travail a résidé dans l’introduction du gradient de protons dans ces équations. Nous avons trouvé que la meilleure manière était de distribuer l’effet du gradient de proton sous forme d’une loi exponentielle sur l’ensemble des paramètres, Vmax et Km pour les substrats et les produits. De cette manière, j’ai montré qu’il était possible de représenter les variations d’oxygène, de ΔΨ et de ΔpH trouvés dans la littérature. De plus, contrairement aux autres modèles, il fut possible de simuler les courbes de seuil observées expérimentalement lors de la titration du flux de respiration par l’inhibiteur d’un complexe respiratoire donné.Ce modèle pourra présenter un très grand intérêt pour comprendre le rôle de mieux en mieux reconnu des mitochondries dans de nombreux processus cellulaires, tels que la production d’espèces réactives de l’oxygène, le vieillissement, le diabète, le cancer, les pathologies mitochondriales etc. comme l’illustrent un certain nombre de prédictions présentées dans ce travail.

Die oxidative Phosphorylierung (OXPHOS) spielt eine zentrale Rolle im Energiestoffwechsel der Zelle. Sie besteht aus der Atmungskette, deren vier Enzymkomplexe einen Protonengradienten über die innere mitochondriale Membran aufbauen, und der ATP-Synthase, die diesen Gradienten zur Phosphorylierung von ADP zu ATP, der zelluläre Energieeinheit, nutzt. In der vorliegenden Arbeit wurde ein thermodynamisch konformes OXPHOS Modell erstellt, welches auf Differentialgleichungen basiert. Dazu wurden Gleichungen entwickelt, welche die Kinetiken jedes OXPHOS-Komplexes über weite Bereiche von Substrat- und Produktkonzentrationen sowie unterschiedlichster Werte des elektrochemischen Gradientens wiedergeben. Zunächst wurden für jeden Komplex der Atmungskette kinetische Messungen in Abwesenheit des Protonengradientens durchgeführt. Für deren Beschreibung erwiesen sich Gleichungen vom Typ Michaelis-Menten als adäquat; hierbei wurden verschiedene Gleichungstypen verglichen. Anschließend wurde der Einfluss des Protonengradientens auf die kinetischen Parameter so modelliert, dass physiologisch sinnvolle Raten in dessen Abhängigkeit erzielt werden konnten. Diese neuen Ratengleichungen wurden schließlich in ein OXPHOS Modell integriert, mit dem sich experimentelle Daten von Sauerstoffverbrauch, elektrischem Potential und pH-Werten sehr gut beschreiben ließen. Weiter konnten Inhibitor-Titrationskurven reproduziert werden, welche den Sauerstoffverbrauch in Abhängigkeit der relativen Hemmung eines OXPHOS-Komplexes darstellen. Dies zeigt, dass lokale Effekte auf globaler Ebene korrekt wiedergeben werden können. Das hier erarbeitete Modell ist eine solide Basis, um die Rolle der OXPHOS und generell von Mitochondrien eingehend zu untersuchen. Diese werden mit zahlreichen zellulären Vorgängen in Verbindung gebracht: unter anderem mit Diabetes, Krebs und Mitochodriopathien, sowie der Bildung von Sauerstoffradikalen, die im Zusammenhang mit Alterungsprozessen stehen. / Oxidative phosphorylation (OXPHOS) plays a central role in the cellular energy metabolism. It comprises the respiratory chain, consisting of four enzyme complexes that establish a proton gradient over the inner mitochondrial membrane, and the ATP-synthase that uses this electrochemical gradient to phosphorylate ADP to ATP, the cellular energy unit. In this work a thermodynamically consistent OXPHOS model was built based on a set of differential equations. Therefore rate equations were developed that describe the kinetics of each OXPHOS complex over a wide concentration range of substrates and products as well for various values of the electrochemical gradient. In a first step, kinetic measurements on bovine heart submitochondrial particles have been performed in the absence of the proton gradient. An appropriate data description was achieved with Michaelis-Menten like equations; here several types of equations have been compared. The next step consisted in incorporating the proton gradient into the rate equations. This was realized by distributing its influence among the kinetic parameters such that reasonable catalytic rates were obtained under physiological conditions. Finally, these new individual kinetic rate expressions for the OXPHOS complexes were integrated in a global model of oxidative phosphorylation. This new model could fit interrelated data of oxygen consumption, the transmembrane potential and the redox state of electron carriers. Furthermore, it could well reproduce flux inhibitor titration curves, which validates its global responses to local perturbations. This model is a solid basis for analyzing the role of OXPHOS and mitochondria in detail. They have been linked to various cellular processes like diabetes, cancer, mitochondrial disorders, but also to the production of reactive oxygen species, which are supposed to be involved in aging.

Nous avons étudié les relations entre les différentes voies du métabolisme énergétique lors de la mise en place des effets Crabtree et Warburg. L’effet du glucose sur le métabolisme énergétique de S. cerevisiae se traduit dans un premier temps par une inhibition cinétique du métabolisme oxydatif (effet Crabtree). Après l’ajout de glucose aux cellules, nous avons mis en évidence l’accumulation d’un intermédiaire de la glycolyse, le F1,6bP. Ceci induit une diminution drastique du rapport G6P/F1,6bP. Or, il a été montré que le G6P stimule et le F1,6bP inhibe l’activité de la chaine respiratoire mitochondriale « in-situ ». L’utilisation de mutants et la modulation de ce rapport nous a permis de montrer que l’induction de l’effet Crabtree chez la levure Saccharomyces cerevisiae est dû à une diminution du rapport G6P/F1,6bP. Parallèlement, le glucose induit un réarrangement génétique qui à terme conduit à un effet Warburg. Nous avons mis en évidence une diminution, au cours du temps du contenu mitochondrial par effet de dilution, suite à un arrêt de la biogenèse mitochondriale (répression de HAP4). Nous avons pu montrer que cette diminution quantitative des OXPHOS est sans effet sur la synthèse d’ATP cellulaire. Ceci est dû à une augmentation du flux de synthèse d’ATP glycolytique. L’utilisation de mutants HAP4", nous a permis de montrer qu’il n’y a pas de lien simple entre prolifération et répression des OXPHOS. Bien que le flux glycolytique diminue dans les conditions de maintien des OXPHOS, ceci est sans effet notoire sur la vitesse de prolifération. Ceci est un rare exemple d’une situation biologique ou l’on observe un découplage entre métabolisme énergétique et prolifération. / We used the yeast Crabtree (+) model to study the relationships between the energy metabolism pathways during the implementation of the Warburg effect. The effect of glucose on S. cerevisiae energetic metabolism results initially in a kinetic inhibition of the oxidative metabolism (Crabtree effect). Rapidly after the addition of glucose, we found an accumulation of F1, 6bP. This induces a drastic reduction in the ratio G6P / F1,6bP. Moreover, it has been shown that G6P stimulates and F1,6bP inhibits the activity of the respiratory chain "in-vitro". Mutants and the modulation of this ratio allowed us to show that the induction of the Crabtree effect is due to a decrease in the G6P / F1,6bP ratio. In parallel with the implementation of the Crabtree effect, glucose induces a genetic rearrangement that leads to a Warburg effect. We showed a decrease over time of mitochondrial enzymatic equipment by dilution effect, due to a halt of mitochondrial biogenesis (transcriptional repression of HAP4). We have been able to show that this decrease in respiratory capacity has no effect on the cellular capacity for ATP synthesis. This is due to the increase in glycolytic ATP synthesis flux. Furthermore, the use of mutants where there is no repression of mitochondrial metabolism upon glucose addition allowed us to show that there is no simple link between OXPHOS activity and cell proliferation. i.e. Mitochondrial metabolism repression/high glycolytic flux is not mandatory to allow a rapid cell proliferation. This is a rare example where energetic metabolism and cell proliferation are uncoupled.

No description available.

Acidithiobacillus ferrooxidans (A. f.) est un modèle bactérien parfaitement adapté à l’étude de la survie en milieu acide. Si plusieurs métalloprotéines ont été identifiées et caractérisées d’un point de vue biochimique, le fonctionnement de la chaîne respiratoire couplant l’oxydation du Fe(II) à la réduction de l’oxygène dans cet organisme n’est pas élucidée. Au cours de ce travail de thèse, après avoir optimisé les conditions de croissance de la bactérie et de production des protéines redox impliquées, nous avons reconstitué sur interface électrochimique une partie de la chaîne respiratoire d’A. f. dans le but de déterminer étape par étape le chemin de transfert d’électrons (TE). Notre attention s’est portée sur trois protéines qui interagissent dans la chaîne respiratoire: la cytochrome c oxidase (CcO), la cuprédoxine AcoP, qui copurifie avec la CcO mais de fonction inconnue, et un cytochrome dihémique (Cyt c4) proposé comme interagissant avec la CcO. La mise en évidence, puis la quantification d’un TE intermoléculaire entre le Cyt c4 et AcoP, puis entre le Cyt c4 et la CcO nous a permis de proposer un rôle pour AcoP et un nouveau chemin de TE vers la CcO. Nous avons ensuite étudié les propriétés électrochimiques de la CcO vis à vis de la réduction catalytique de l’O2, en particulier avec une forte affinité. Nous avons ainsi pu montrer que la CcO de A. f. réduisait l’O2 à des potentiels 500 mV plus anodiques que les CcO neutrophiles par une connexion directe de l’enzyme sur nanomatériaux carbonés. Affinité pour O2 et haut potentiel redox font de cette CcO une enzyme de choix pour développer une nouvelle génération de piles à combustible enzymatique. / Acidithiobacillus ferrooxidans is one of the most studied bacterial models to understand how to survive in an acid environment. Although several metalloproteins have been identified and characterized from a biochemical point of view, the electron transfer pathway (ET) of the respiratory chain coupling the oxidation of ferrous iron with the reduction of oxygen in this organism has not been elucidated.During this thesis work, after having optimized the growth conditions of the bacterium and the production of the redox proteins involved, we reconstituted on the electrochemical interface part of the respiratory chain of A. ferrooxidans for the purpose of determining step by step the ET. Our attention focused on three proteins that interact in the respiratory chain: cytochrome c oxidase (CcO), the cupredoxin AcoP, which co-purifies with CcO but of unknown function, and a cytochrome dihemic (Cyt c4) proposed as interacting with the CcO. The demonstration, then the quantification of an intermolecular ET between the Cyt c4 and AcoP, then between the Cyt c4 and the CcO allowed us to propose a role for AcoP and a new pathway for the ET to the CcO. We then studied the electrochemical properties of CcO with respect to the catalytic reduction of O2. We have demonstrated the strong affinity of this oxidase for O2. We have established the chemical functions required to obtain a direct wiring of the enzyme on carbon nanomaterials. This showed that A. ferrooxidans CcO reduced O2 at potentials 500 mV more anodic than neutrophilic CcOs. Affinity for O2 and high redox potential make this CcO an enzyme of choice to develop a new generation of enzymatic fuel cells.

Diese Arbeit beschäftigt sich mit dem mitochondrialen Sco1-Protein der Hefe Saccharomyces cerevisiae sowie mit weiteren Vertretern der Sco-Proteinfamilie. Sco1p ist essenziell für die Assemblierung der Cytochrom c Oxidase (COX), dem terminalen Komplex der Atmungskette. Aufgrund von genetischen Daten wurde angenommen, dass es an der Insertion von Cu-Ionen in den COX-Komplex beteiligt ist. Dabei existieren zwei unterschiedliche Vorstellungen über seine Wirkweise: Einerseits könnte Sco1p als Cu-Chaperon selbst Cu-Ionen binden und anschließend auf die Cu-tragenden COX-Untereinheiten Cox1p und/oder Cox2p übertragen. Andererseits könnte es als Disulfidreduktase die in die Cu-Bindung involvierten Cysteinreste von Cox2p reduzieren und somit die Voraussetzung für eine Cu-Anheftung an Cox2p schaffen. In beiden Fällen wird den unter den Sco-Proteinen konservierten Aminosäuren Cystein(148), Cystein(152) und Histidin(239) eine Schlüsselrolle zugedacht. Es wurde gezeigt, dass diese Aminosäuren tatsächlich essenziell für die Funktion von Sco1p sind. Die Daten dieser Arbeit sprechen dafür, dass Sco1p als Cu-Chaperon fungiert: Sco1p zeigt keine Aktivität als Disulfidreduktase. Außerdem interagiert Sco1p mit Cox17p - dem Protein, das Cu-Ionen in die Mitochondrien importiert - und geht mit Cox2p eine Wechselwirkung ein. Im Rahmen der Interaktionsanalysen wurde weiterhin gezeigt, dass Sco1p homomere Komplexe ausbildet. Ein weiterer Schwerpunkt dieser Arbeit lag in Untersuchungen zum homologen Sco2p aus Saccharomyces cerevisiae, das im Gegensatz zu Sco1p nicht essenziell für eine funkionsfähige COX ist. Trotz seiner großen Ähnlichkeit ist Sco2p nicht in der Lage, die Funktion von Sco1p zu erfüllen. Im Rahmen dieser Arbeit konnt aber demonstriert werden, dass Sco2p zumindest teilweise Sco1p ersetzen kann. Somit kann für beide Proteine angenommen werden, dass sie überlappende Funktionen besitzen. Übereinstimmend wurde nachgewiesen, dass Sco2p - wie Sco1p - in der Lage ist, mit Cox17p und mit Cox2p zu interagieren und außerdem heteromere Komplexe mit Sco1p formiert. Es wurde ein Modell zur Wirkweise von Sco1p und Sco2p entwickelt.

This study investigates the connection between different pathophysiological processes in mitochondria and psychopathological symptoms in patients with bipolar disorder. Changes in activity of selected components of the respiratory chain and overall respiratory rate of mitochondria were analyzed in patients with bipolar disorder when compared to healthy controls. Diagnostic scales and questionnaires, high-resolution respirometry, radiochemical and spectroscopic methods were used. 37 patients with a diagnosis of bipolar disorder (F31) and 21 healthy volunteers were involved in the study. Statistical analysis included the methods of parametric and nonparametric analysis, factor analysis, one-way analysis of variance and linear regression analysis. Obtained results revealed that cellular energetics plays a great role in the pathophysiology of bipolar disorder. There was a mild difference between different mitochondrial enzymes activity in patients within manic phases and depressive phases of the disease. Changes in mitochondrial respiration in patients with BD as compared to healthy controls were also shown. Mitochondrial respiration indexes for patients with BD in remission as compared to healthy controls were altered in accordance with the previous phase of the disease. Association between the...

Le domaine de la bioénérgétique traîte de la circulation et de la transformation de l’énergie dans et entre des organismes et leur environnement. Dans ce manuscrit de thèse, la respiration cellulaire et plus particulièrement la première enzyme de la chaîne respiratoire, la NADH:ubiquinone oxidoreductase (Complexe I) ont été étudiées, dans l’objectif de clarifier sa fonction et son implication dans certaines maladies. Dans une première partie, la création d’une sonde impliquant l’enzyme immobilisée de façon biomimétique est décrite. La caractérisation de ce système est effectuée via spectroscopie infrarouge par exaltation de surface (SEIRAS) couplée à de l’électrochimie. Sa réponse à l’ajout de substrats et d’inhibiteurs est ensuite présentée. Dans une seconde partie, l’interaction du Complexe I avec des lipides et des inhibiteurs (Zn2+ et NADH-OH) ainsi que le rôle d’une Tyrosine située au site de fixation du NADH ont été étudiés par spectroscopies IR et UV-Vis différentielles induites par électrochimie. L’exploration des résultats obtenus sous un angle structural a finalement permis de proposer un modèle pour le mécanisme de couplage entre la réduction d’ubiquinone et le pompage de protons par le Complexe I. / The field of bioenergetics deals with the flow and transformation of energy within and between living organisms and their environment. The work presented in this thesis report focuses on cellular respiration and more specifically on the first enzyme of the respiratory chain, NADH:ubiquinone oxidoreductase (Complex I). This was done to clarify details about its function and its implication in disease. First, the creation of a sensor involving the biomimetically immobilized enzyme is presented and probed through a combination of surface enhanced infrared absorption spectroscopy (SEIRAS) and electrochemistry. This sensor is then tested against different substrates and inhibitors. In a second part, the interaction of Complex I with lipids, inhibitors (Zn2+ and NADH-OH) and the role of a Tyrosine residue situated in the NADH binding pocket are investigated through electrochemically induced UV-Vis and FTIR difference spectroscopies. The results gathered through these experiments are then explored under a structural perspective and a coupling mechanism between quinone reduction and proton translocation by Complex I is proposed.

Amino acids are the building blocks of proteins, and the use of balanced diets with essential amino acids increases animal performance favored by the increased expression of specific genes. The experiment was conducted in Advanced Laboratory of Poultry of Department Animal Science of the Federal University of Sergipe. Two hundred and fifty two male broilers chickens were used up to 42 days old. For the first seven days of age the birds were reared in broiler house with floor covered with wood shavings. After this age, the birds were distributed in metabolism cages in randomized design with four treatments and seven replicates of nine birds per cage. The treatments consisted of diets with containing four levels of lysine, 1.016; 1.099; 1.182 and 1.265 % for the period from 8 to 21 days of age and 0.923; 0.998; 1.073 and 1.148 % for the period from 22 to 42 days, being isoenergetics and isonitrogenous diets. The characteristics evaluated were: weight gain, feed intake, feed conversion; carcass yield and parts; deposition of nutrients and gene expression in the Pectoralis major muscle. Four birds per treatment were slaughtered by cervical dislocation at 21, 35 and 42 days of ages for removal of five grams of muscle tissue and amplification of the cDNA using primers specific for the target genes. Expression was analyzed using the Real-Time Polymerase Chain Reaction (RT-qPCR). Weight gain and feed conversion were influenced by increasing in the dietary digestible lysine. For carcass yield and parts, the breast was influenced by the increase of dietary lysine. It was found that there was an increase in protein daily gain when used 1.265 and 1.148 % digestible lysine in the different ages. For gene expression, there was no significant effect of lysine on the evaluated genes (ND1, ND2, COX I, COX II, COX III, Cyt b e ATP6). Based on the information concluded that adjusted levels of digestible lysine for phases 8 to 21 and 22 to 42 days of age provide higher performance and body protein deposition. However, increasing levels of lysine does not influence the expression of the electron transport chain genes and oxidative phosphorylation in the Pectoralis major muscle of male broilers. / Os aminoácidos são constituintes básicos das proteínas e o uso de rações equilibradas em aminoácidos essenciais contribui para o aumento do desempenho animal, favorecido possivelmente pelo aumento da expressão de genes específicos. O experimento foi desenvolvido no Núcleo de Estudos Avançados em Avicultura do Departamento de Zootecnia da Universidade Federal de Sergipe. Foram utilizados 252 frangos de corte, machos, da linhagem Cobb 500 até 42 dias de idade. Até os sete dias de idade as aves foram criadas em galpão de alvenaria com piso coberto com maravalha e após essa idade, aves foram distribuídas em gaiolas de metabolismo num delineamento inteiramente casualizado em quatro tratamentos com sete repetições de nove aves por unidade experimental. Os tratamentos consistiram de rações com quatro níveis de lisina digestíveis, 1,016; 1,099; 1,182 e 1,265% para a fase de 8 a 21 dias e 0,923; 0,998; 1,073 e 1,148% para a fase de 22 a 42 dias de idade, sendo isoenergéticas e isoproteicas. As características avaliadas foram: ganho de peso, consumo de ração, conversão alimentar; rendimento de carcaça e partes; deposição de nutrientes e expressão de genes no músculo Pectoralis major. Quatro aves por tratamento foram abatidas aos 21, 35 e 42 dias de idade para retirada de cinco gramas de tecido muscular e amplificação do cDNA, com uso dos primers específicos para os genes alvos. A expressão foi analisada usando a Reação em Cadeia da Polimerase em tempo real (RT-qPCR). O ganho de peso e a conversão alimentar foram influenciados positivamente pelo aumento da lisina digestível nas rações. Para rendimento de carcaça e partes, o peito foi a única variável influenciada pelo aumento de lisina nas rações. Nas diferentes idades de criação, verificou-se que houve acréscimo no ganho diário proteico quando utilizados 1,265 e 1,148% de lisina digestível. Para expressão gênica, não foi observado efeito significativo da lisina sobre os genes avaliados (ND1, ND2, COX I, COX II, COX III, Cyt b e ATP6). Baseado nas informações obtidas conclui-se que rações ajustadas com níveis de lisina digestível para as fases de 8 a 21 e 22 a 42 dias, proporcionam maior desempenho e deposição de proteína corporal. Porém, não influencia na expressão de genes da cadeia transportadora de elétrons e fosforilação oxidativa no músculo Pectoralis major de frangos de corte machos.