In cellular respiration, electron transfer from the breakdown of foodstuff is coupled to the formation of an electrochemical proton gradient. This is accomplished through proton translocation by respiratory complexes, and the proton gradient is subsequently used e.g. to drive ATP production. Consequently, proton- and electron-transfer reactions through the hydrophobic interior of membrane proteins are central to cellular respiration. In this thesis, proton- and electron transfer through an aa3-type terminal oxidase, cytochrome c oxidase (CytcO) from Rhodobacter sphaeroides, have been studied with the aim of understanding the molecular proton-transfer machinery of this proton pump. In the catalytic site of CytcO the electrons combine with protons and the terminal electron acceptor O2 to form water in an exergonic reaction that drives proton pumping. Therefore, CytcO must transfer both protons that are pumped and protons for the oxygen chemistry through its interior. This is done through its two proton-transfer pathways, termed the D pathway and the K pathway. Our studies have shown that the protons pumped during oxidation of CytcO are taken through the D pathway, and that this process does not require a functional K pathway. Furthermore, our data suggests that the K pathway is used for charge compensation of electron transfer to the catalytic site, but only in the A2 → P3 state transition. Our data also show that the water molecules identified in the crystal structures of CytcO play an important role in proton transfer through the D pathway. Finally, the effects of liposome reconstitution of CytcO on D-pathway proton transfer were investigated. The results suggest that the membrane modulates the rates of proton transfer through the D pathway, and also influences the extent of electron transfer between redox-active sites CuA and heme a.

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transport chain, couples proton pumping to the reduction of dioxygen into water. The coupling mechanism remains to be elucidated. Previous studies have identified several mutations within CcO's primary proton uptake pathway (the D-channel) that decouple proton pumping from redox activity. Here, I examine the molecular basis for decoupling in single and double mutants of highly conserved residues, D132 and N139, in order to gain insight into the coupling mechanism. In particular, I use molecular dynamics and free energy simulations of a new, unconstrained model of bacterial CcO embedded in a solvated lipid bilayer to investigate how such mutants affect functional hydration and ionic selectivity in the D-channel. Results support earlier mechanistic insights obtained in our laboratory from simplified molecular models and predict a new, testable hypothesis by which cations such as K+ may inhibit proton pumping in charged mutants of N139.

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transport chain, couples proton pumping to the reduction of dioxygen into water. The coupling mechanism remains to be elucidated. Previous studies have identified several mutations within CcO's primary proton uptake pathway (the D-channel) that decouple proton pumping from redox activity. Here, I examine the molecular basis for decoupling in single and double mutants of highly conserved residues, D132 and N139, in order to gain insight into the coupling mechanism. In particular, I use molecular dynamics and free energy simulations of a new, unconstrained model of bacterial CcO embedded in a solvated lipid bilayer to investigate how such mutants affect functional hydration and ionic selectivity in the D-channel. Results support earlier mechanistic insights obtained in our laboratory from simplified molecular models and predict a new, testable hypothesis by which cations such as K+ may inhibit proton pumping in charged mutants of N139.

In neurons, mitochondrial quantity and basal cellular respiration are maintained with age, but alterations in other key functions and quantities make these cells susceptible to the pathology of age-related neurodegenerative disease. We observed age-related decreases in cytochrome C, cardiolipin, cytochrome C oxidase (CCO) function, and glutamate response that render cells less capable of responding to stress. Rescue experiments showed that estrogen is a promising treatment in restoring neuron function with age. After finding key differences in CCO, we examined the electron transport chain more closely and found age-related deficits in quantity or function for each individual complex. Our experiments support a lack of endogenous substrates or a failure of upstream complexes to transport electrons to complex IV with age, ultimately leading to age-related neurodegeneration. Reactive oxygen species production may add to the problem by degrading macromolecules such as nucleic acid, cardiolipin, and proteins. Increased ROS may also lead to a redox imbalance in the neuron, reducing the potential for energy production. Also, epigenetic controls such as DNA methylation, histone acetylation ubiquitination and phosphorylation that persist in culture independent of aging hormone levels, vasculature, and immune system may be partly responsible for the observed age-related deficiencies as has been previously observed in aging human muscle (Ronn et al., 2008). This compelling cumulative evidence suggests an age-related deficiency in electron transport via quinones from complexes I to III, and age-related deficiencies in substrates, cofactors, and quantity or function for complex IV. These studies add to the growing body of evidence that dysfunction in the enzyme complexes of the electron transport chain lead to neurodegeneration in senescence-related diseases. In an attempt to integrate our age-related findings with Alzheimer's Disease (AD) pathology, we sequentially isolated the electron transport chain complexes using selective mitochondrial inhibitors in cortical neurons removed from the 3xTg-AD mouse model, which harbors mutations in the PS1, APPSwe and tauP301L genes and follows the proposed temporal development of human AD pathology (Oddo et al., 2003a; 2003b). Overall, we did not detect 3xTg-AD cortical neuron deficits at the four electron transport complexes of mitochondria or in NAD(P)H oxidase (NOX), an extramitochondrial oxygen consumer and regulator of NAD(P)+/NAD(P)H homeostasis (Morre et al., 2000).

No description available.

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.

Dans cette thèse, le processus d'assemblage ainsi que la maturation du cytochrome c oxydase de type cbb3 (cbb3-Cox) ont été étudiés dans la proteobactérie phototrophique pourpe non soufrée Rhodobacter capsulatus. R. capsulatus contient une chaîne de transfert d'électrons très ramifiée et represente un modèle d’organisme très utilisé dans l'étude des processus respiratoires et photosynthétiques.Les cbb3-Coxs spécifiques des bactéries représentent la deuxième catégorie la plus abondante des cytochromes c oxydases après le type Cox-aa3, mais n'ont jusqu'à présent pas été étudiées en détail. Récemment, la première structure cristalline cbb3-Cox de P. stutzeri a été obtenue, fournissant ainsi une avancée majeure invitant à des etudes plus détaillées sur le mécanisme catalytique et le processus d'assemblage. Les études sur les procédés d'assemblage et de maturation sont d'une très grande importance en raison du fait que de nombreux agents pathogènes humains tels que Helicobacter pylori, Neisseria meningitidis ou Campylobacter jejuni utilisent ce type de Cox, ce qui par conséquent pourrait amener a développer une interessante cible thérapeutique.Cbb3-Cox dans R. capsulatus est encodé par le gène opéron ccoNOQP codant quatre protéines membranaires constitutives de cbb3-Cox. CcoP et CcoO sont des cytochromes de type c, contenant des motifs périplasmiques fixés à l’hème. CcoN est la sous-unité centrale catalytique contenant deux molécules d’hèmes de type b et un ion cuivre. L’étude de la distribution de l’ion Cu à la sous-unité CcoN et l'assemblage des quatre sous-unités dans le complexe actif cbb3-Cox complexe sont les thèmes principaux de ce travail.Ici, le rôle du facteur d'assemblage putatif CcoH, sa structure et son interaction avec cbb3-Cox ont été étudiés en détail. CcoH est une petite protéine membranaire codé dans le groupe de gènes ccoGHIS situé à proximité des gènes codant cbb3-Cox. L'analyse in vivo de la formation de cbb3-Cox dans une souche ne contenant pas le facteur CcoH a montré une absence totale de cbb3-Cox. De même, la stabilité du facteur CcoH a été considérablement altérée dans une souche avec deletion du gene ccoNOQP. La dépendance mutuelle des deux protéines suggère leur interaction directe, ce qui a été confirmé par la photoréticulation directe de CcoH à la sous-unité CcoN, l’immunodétection de CcoH dans les cbb3-Cox complexes sur gels Blue Native, la co-purification par marquage CcoH-cbb3-Cox et le marquage radioactive in vitro des complexes cbb3-Cox avec CcoH.[...] / In this thesis, the assembly and maturation process of the cbb3-type cytochrome c oxidase (cbb3-Cox) was studied in the purple-non-sulphur phototrophic α-proteobacterium Rhodobacter capsulatus. R. capsulatus contains a highly branched electron-transfer chain and is a well studied model organism for investigating respiratory and photosynthetic processes.The bacteria-specific cbb3-Coxs represent the second most abundant class of cytochrome c oxidases after the aa3-type Cox, but have so far not been investigated in much detail. Recently, the first crystal structure of cbb3-Cox from P. stutzeri was obtained, providing a major breakthrough and inviting detailed studies on the catalytic mechanism and the assembly process. Studies on the assembly and maturation processes are of wide significance due to the fact that many human pathogens like Helicobacter pylori, Neisseria meningitides or Campylobacter jejuni use this type of Cox and it therefore might develop into an attractive drug-target. cbb3-Cox in R. capsulatus is encoded by the ccoNOQP gene operon which codes for four membrane proteins constituting cbb3-Cox. CcoP and CcoO are c-type cytochromes, containing periplasmic heme-binding motifs. CcoN is the central catalytic subunit which contains two b-type hemes and a copper ion. Investigating the delivery of Cu to the CcoN subunit and the assembly of all four subunits into the active cbb3-Cox complex were the main topics of this work. Here the role of the putative assembly factor CcoH, its structure and interaction with cbb3-Cox was investigated in detail. CcoH is a small membrane protein encoded in the ccoGHIS gene cluster located adjacent to the genes coding for cbb3-Cox. In vivo analysis of cbb3-Cox formation in a strain lacking ccoH showed the total absence of cbb3-Cox. Likewise, the stability of CcoH was drastically impaired in a ccoNOQP deletion strain. The mutual dependency of both proteins suggested their direct interaction, which was confirmed by site-directed photocrosslinking of CcoH to the CcoN subunit, by immunodetection of CcoH in cbb3-Cox complexes on Blue Native gels, by CcoH-cbb3-Cox co-purification and by in vitro labelling of cbb3-Cox complexes with radioactively labelled CcoH.[...]

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

Maintaining energy organismal homeostasis under changing physiological and environmental conditions is vital, and requires constant adjustments of the energy metabolism. Central to meeting energy demands is the regulation of mitochondrial oxidative capacity. When demands increase, animals can increase mitochondrial content/enzymes, known as mitochondrial biogenesis. Central to mammalian mitochondrial biogenesis is the transcriptional master regulator PPARγ (peroxisome proliferator-activated receptor γ) coactivator-1α (PGC-1α), and the network of DNA-binding proteins it coactivates (e.g. nuclear respiratory factor 1 and 2 [NRF-1, NRF-2], estrogen-related receptor α [ERRα], thyroid receptor α [TRα-1], retinoid X receptor α [RXRα]). However, the mechanisms by which mitochondrial content in lower vertebrates such as fish is controlled are less studied. In my study I investigate underlying mechanisms of the phenomenon that many fish species alter mitochondrial enzyme activities, such as cytochrome c oxidase (COX) in response to low temperatures. In particular, I investigated (i) if the phenomenon of mitochondrial biogenesis during cold-acclimation is related to fish phylogeny, (ii) what role PGC-1α and other transcription factors play in mitochondrial biogenesis in fish, and (iii) if mRNA decay rates are important in the transcriptional control of a multimeric protein like COX. This study shows that mitochondrial biogenesis does not follow a phylogenetic pattern: while distantly related species displayed the same response to low temperatures, closely related species showed opposite responses. In species exhibiting mitochondrial biogenesis, little evidence was found for PGC-1α as a master regulator, whereas NRF-1 is supported to be an important regulator in mitochondrial biogenesis in fish. Further, there was little support for other transcription factors (NRF-2, ERRα, TRα-1, RXRα) to be part of the regulatory network. Lastly, results on the post-transcriptional control mechanism of mRNA decay indicate that this mechanism is important in the regulation of COX under mitochondrial biogenesis: it accounts for up to 30% of the change in subunit transcript levels. In summary, there is no simple temperature-dependent mitochondrial response ubiquitous in fish. Further, the pathways controlling mitochondrial content in fish differ from mammals in the important master regulator PGC-1α, however, NRF-1 is important in regulating cold-induced mitochondrial biogenesis in fish. Lastly, COX subunit mRNA decay rates seem to have a part in controlling COX amounts during mitochondrial biogenesis. / Thesis (Ph.D, Biology) -- Queen's University, 2013-10-21 09:53:59.46

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