O objetivo deste trabalho foi desenvolver biosensores de membranas baseados na técnica de Ressonância Plasmônica de Superfície (SPR) e aplicá-los no estudo da interação do citocromo c (cit c) com modelos de membranas. SPR é uma técnica ótica, que através de medidas de variações de índice de refração (n) próximas a uma interface mensura com alta sensibilidade a adsorção ou ligação de moléculas. Inicialmente desenvolvemos um sistema de gradiente de fluxo acoplado ao SPR, denominado FIG-SPR, e demonstramos a determinação automatizada da variação de n em função da concentração (dn/dC) de diferentes compostos e biopolímeros. O desenvolvimento dos biosensores de membranas iniciou-se com o estudo dos fatores que afetam a formação de uma membrana de bicamada híbrida (HBM). HBMs são compostas de uma monocamada de alcanotiol adsorvida sobre o ouro, e sobre esta uma camada fosfolipídica. A formação da HBM depende da fusão de vesículas em superfícies hidrofóbicas e que não é bem compreendido no nível molecular. Nossos estudos mostraram que na presença de cálcio e espermina a formação da HBM é favorecida, de tal forma que a monocamada de fosfolipídio alcança valores de espessura próximos àqueles previstos, cerca de 20 Å\'. Além disso, mostramos que em soluções de baixa força iônica a camada lipídica não é homogênea. Demonstramos também que a presença de cálcio na concentração 150 mM diminui o tempo de formação da monocamada lipídica cerca de 14 vezes quando comparado ao tempo indicado na literatura. A homogeneidade da HBM e a carga superficial da mesma foram verificadas com a adsorção e a dissociação de cit c e de albumina bovina (BSA). Utilizando HBMs de composição lipídica variada demonstramos a adsorção e a dissociação de cit c induzida por cálcio em HBMs mistas, incluindo um modelo mimético da membrana mitocondrial interna (IMM) constituído de fosfatidilcolina, fosfatidiletanolamina e cardiolipina (PC/PE/CL) na proporção (4,5:3,5:2,0). Demonstramos que a adsorção de cit c nativo segue um perfil cooperativo e padrões esperados de variação de afinidade e cooperatividade em pHs 6,8, 7,4 e 8,0. Um modelo matemático foi desenvolvido para tratar as curvas de ligação de cit c, que é uma adaptação do modelo de Hill para adsorção de proteínas em superfícies. Os resultados de SPR juntamente com dados obtidos por Microscopia de Força Atômica (AFM) sugerem que a ligação cooperativa de cit c com HBM ocorre devido à reorganização das moléculas de CL e formação de domínios fosfolipídicos. O tratamento dos resultados cinéticos da dissociação de cit c por cálcio indica a existência de duas constantes de velocidade de dissociação (kd), sendo a primeira constante (kd1) relacionada à perda das interações eletrostáticas entre a proteína e a HBM, e a segunda (kd2) à perda das interações hidrofóbicas. Além disso, a dissociação do cit c do modelo estudado requer uma concentração mínima de cálcio de 30 µM para se tornar significativa. O estudo da interação entre moléculas de cit c foto-oxidadas (citc405) e a HBM de PC/PE/CL sugerem que ela ocorre com menor afinidade, nos três pHs estudados, se comparados aos resultados com cit c nativo. Além disso, nossos resultados sugerem que o citc405 não é facilmente dissociado por cálcio devido à perda da cooperatividade na interação. Possíveis implicações em eventos celulares destas descobertas, como a liberação do cit c da IMM e a iniciação da apoptose, são discutidas / The aim of this work was to develop membrane biosensors based on Surface Plasmon Resonance (SPR) and to apply them to study the interactions between cytochrome c (cyt c) and model membranes. SPR is an optical technique that provides high-sensitivity measurements of refractive index (n), allowing the characterization of the adsorption and desorption of molecules near interfaces. Initially we developed a flow gradient system connected to SPR, which was called FIG-SPR, and demonstrated the automated determination of the concentration gradient of refractive index (dn/dC) of different materials and biopolymers. The development of the membrane biosensors was initiated by studying the factors that affect the formation of a hybrid bilayer membrane (HBM). HBMs are composed of two monolayers: an alcanethiol monolayer adsorbed on gold over which is adsorbed a layer of phospholipids. The formation of an HBM depends on the fusion of phospholipid vesicles on hydrophobic surfaces, a process that is not well understood at the molecular level. Our results showed that in the presence of calcium and spermine the complete formation of an HBM is facilitated, i.e., the phospholipid monolayer reaches the expected thickness of about 20Å\'. However, in low ionic strength solutions the lipid layer that is formed is not homogeneous. We have also demonstrated that in the presence of 150 mM of calcium the time necessary for the formation of the lipid monolayer is reduced 14 times when compared to the times suggested in the literature. The homogeneity of the HBM and its superficial charge were verified with the adsorption and desorption of cyt c and bovine serum albumine (BSA). The adsorption and desorption of cyt c in different HBMs were studied including a model of the internal mitochondrial membrane (IMM), which is made of phosphatidylcholine, phosphatidylethanolamine and cardiolipin (PC/PE/CL) in the ratio (4,5: 3,5: 2,0). We demonstrated that the adsorption of native cyt c follows a cooperative profile showing expected changes in affinity and cooperativity in different solution pHs of 6,8, 7,4 and 8,0. A mathematical model, which is an adaptation of the Hill model for adsorption of proteins in surfaces, was developed to treat the binding curves of cyt c. The results of SPR together with those obtained by Atomic Force Microscopy (AFM) suggested that the cooperative binding of cyt c in HBMs occurs due to the reorganization of CL molecules and formation of phospholipid domains. The kinetic results of the dissociation of cyt c induced by calcium indicates the existence of two velocity constants (kd), being the larger (kd1) related to the dissociation of cyt c interacting electrostatically with the HBM, and the smaller (kd2) related to the dissociation of cyt c interacting hydrophobically with the HBM. Moreover, the dissociation of cyt c from the HBM requires a minimum calcium concentration of 30 µM. The study of the interaction between photo-oxidized cyt c molecules (cytc405) and the PC/PE/CL HBM suggests that it occurs with smaller affinity when compared with the results obtained with the native cyt c. Moreover, cytc405 is not easily dissociated by calcium due to the loss of the interaction cooperativity with the HBM. Possible implications of these discoveries in cellular events, such as the release of cyt c from the IMM and the initiation of apoptosis, are discussed

Oxygen and nitric oxide reductases are enzymes found in aerobic and anaerobic respiration, respectively. Both enzyme groups belong to the superfamily of Heme-Copper Oxidases, which is further divided into several subgroups: oxygen-reducing enzymes into A-, B- and C-type and nitric oxide reductases into qNORs and cNORs. Oxygen reducing enzymes use the energy released from oxygen reduction to take up electrons and protons from different sides of the membrane. Additionally, protons are pumped. These processes produce a membrane potential, which is used by the ATP-synthase to produce ATP, the universal energy currency of the cell. Nitric oxide reductases are not known to conserve the energy from nitric oxide reduction, although the reaction is highly exergonic. Here, the detailed mechanism of a B-type oxidase is studied with special interest in an element involved in proton pumping (proton loading site, PLS). The study supports the hypothesis that the PLS is protonated in one and deprotonated in the consecutive step of the oxidative catalytic cycle, and that a proton is pumped during the final oxidation phase. It further strengthens the previous suggestion that the PLS is a cluster instead of a single residue or heme propionate. Additionally, it is proposed that the residue Asp372, which is in vicinity of the heme a3 propionates previously suggested as PLS, is part of this cluster. In another study, we show that the Glu15II at the entry of the proton pathway in the B-type oxidase is the only crucial residue for proton uptake, while Tyr248 is or is close to the internal proton donor responsible for coupling proton pumping to oxygen reduction. The thesis also includes studies on the mechanism and electrogenicity of qNOR. We show that there is a difference in the proton-uptake reaction between qNOR and the non-electrogenic homolog cNOR, hinting at a different reaction mechanism. Further, studies on a qNOR from a different host showed that qNOR is indeed electrogenic. This surprising result opens up new discussions on the evolution of oxygen and nitric oxide reductases, and about how energy conservation can be achieved. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

Ubiquinol-cytochrome c reductase (complex III) is a central component of the respiratory chain of the inner mitochondrial membrane. It transfers electrons from reduced ubiquinone to ferricytochrome c. Correctly assembled and functional complex III is an essential prerequisite for oxidative energy metabolism. Complex III deficiency has been reported to be associated with several neurodegenerative diseases. Formation and assembly of complex III requires a multitude of specific nuclearly encoded proteins. For example, gene specific translational activators for cytochrome b synthesis as well as three non-subunit proteins, which are important for assembly and/or stability have been detected. The role of Bcs1p in assembly of Rieske FeS protein and Qcr10p into complex III has been clasified recently. The role of the two putative chaperones, Cbp3p and Cbp4p, is not known. In spite of the similar phenotype of cbp3D and cbp4D strains, that suggests the role of both proteins in the same step of complex III assembly, we were able for the first time to demonstrate differences on the molecular level between both deletion mutants. We show by BN-PAGE that cbp3D and cbp4D mutants are disturbed in complex III assembly and accumulate intermediate-sized forms of the complex. Moreover deletion of CBP3 interferes with the formation of complex III/IV supracomplexes. Our studies show that Cbp3p and Cbp4p interact and are present in high molecular weight complexes, some of which might represent intermediates of complex III assembly. Overexpression of Cbp4p cannot substitute for the function of Cbp3p, but high level expression of Cbp3p can partially compensate for the lack of Cbp4p. Because lipids play an important role for complex III assembly and stability, we analysed the mitochondrial lipid composition of cbp3D and cbp4D mutants. Our data show that mitochondria of both mutants exhibit a wild type-like lipid composition, that favors the idea that Cbp3p and Cbp4p are specific assembly factors for complex III rather than components of the mitochondrial lipid metabolism. By complementation studies we have shown that Cbp3 proteins of S. cerevisiae, S. pombe and human are (partially) functional homologues. A yeast model based on chimeric constructs of S. cerevisiae and human proteins was constructed, which allows to test the pathogenicity of human mutations. To define the role/s of Cbp3p and Cbp4p in the assembly pathway of complex III, interactions of selected subunits with both assembly factors were analysed by TAP- or co-immunoprecipitation. Based on the results of Cbp3p and Cbp4p topologies, BN-PAGE analysis of null mutant strains and interaction studies a model for complex III assembly and the roles of Cbp3p and Cbp4p in this process are proposed. I present a hypothesis, according to which Cbp3p and Cbp4p form a ?scaffold? for the assembly of all three putative sub-complexes, may act independently in the first steps of bc1 complex assembly (e. g. the formation of sub-complexes) and interact together to assist the final assembly of sub-complexes into a mature enzyme. / Der Ubiquinol-Cytochrom c Reductase (Komplex III) ist eine zentrale Komponente der Atmungskette der inneren Mitochondrienmembran. Er transferiert Elektronen von reduziertem Ubiquinon auf Ferricytochrom c. Der korrekt assemblierte und funktionale Komplex III ist eine essenzielle Voraussetzung für den oxidativen Energiemetabolismus. Komplex III Defizienz ist assoziiert mit verschiedenen neurodegenerativen Krankheiten...

In der vorliegenden Arbeit sind unterschiedliche Kopplungsstrategien des natürlichen Photosystems I (PSI) aus Cyanobakterium Thermosynechococcus elongatus mit verschiedenen Elektrodenoberflächen sowie Interaktion mit Nanomaterialien und Enzymen bearbeitet worden. Zum einen wurde gezeigt, dass die Immobilisierung des PSI auf modifizierten mehr-wandigen Kohlenstoffnanoröhrchen zur funktionalen Photobiohybridelektrode führt. Dabei wurde das PSI mit der Elektrode elektrisch mit Hilfe eines Redoxproteins, Cytochrom c (cyt c), verknüpft. Das System (PSI-cyt c) wurde auch auf eine dreidimensionale Elektrodenoberfläche des Metaloxids Indiumzinnoxid (eng. ITO) übertragen. Hierbei wurde zusätzlich die TransparenzEigenschaft solcher Oberflächen ausgenutzt. Die Präparation solcher transparenter Elektroden wurde optimiert, um höhere Photoströme zu generieren. Weiterhin wurde eine neue Methode der elektrischen Kontaktierung des PSI mit der Elektrode etabliert. Hierfür wurden Fullerene eingesetzt. Durch erhöhte molekulare Effizienz wurde gezeigt, dass Fullerene effektivere Elektronvermittler zwischen PSI und der Elektrode sind als das cyt c. Zusätzlich wurden im Rahmen dieser Doktorarbeit die photokatalytischen Eigenschaften von PSI mit den biokatalytischen Eigenschaften des Enzyms humane Sulphit Oxidase (hSOx) kombiniert. Hierbei wurde das Enzym als ein alternativer und effizienter Elektronzulieferer für PSI eingesetzt. Ein drittes Protein, das cyt c, fungierte als elektrisches Bindeglied und sicherte die elektrische Kommunikation zwischen den katalytischen Proteinen im System und der Elektrode. Die Komplexität des PSI sowie seine Kommunikation mit anorganischen Nanomaterialien und anderen komplexen Biomolekülen, wie z.B. Enzymen, zeigt ein großes Potential des Einsatzes von PSI-basierter Biohybriden in den Biotechnologien der Zukunft. / In this thesis, different strategies for coupling of the natural complex photosystem I from the cyanobacterium Thermosynechococcus elongatus with different electrode surfaces, and the interaction of PSI with nanomaterials and enzymes has been investigated. First, it was shown that immobilization of PSI on modified multi-walled carbon nanotubes (MWNT) leads to a functional photobiohybrid electrode. Here, PSI has been electrically wired to the electrode via a redox-active protein, cytochrome c (cyt c). The system (PSI-cyt c) has been scaled up to the three-dimensional surface of a metal-oxide, indium tin oxide (ITO). Here, additionally the high transparency property of this material has been exploited. The new preparation procedure of such transparent electrodes has been optimized in order to achieve high pohotocurrents. Furthermore, a new method of electric wiring of the PSI with the electrode has been established. Here, fullerenes have been employed. The high molecular efficiency of such a system proves that fullerenes are more effective wiring agents between the PSI and the electrode as compared to the cyt c. Additionally, in this thesis the photocatalytic property of the PSI has been combined with the biocatalytic property of the enzyme human sulphite oxidase, hSOx. Here, the enzyme has been employed as an alternative electron supplier for PSI. The third protein, cyt c, acted as an electric wiring agent and ensured electric communication between both catalytic proteins of the system and the electrode. The versatility of the PSI as well as its communication with anorganic nanomaterials and biological molecules, e.g. such as enzymes, shows a great potential for use of PSI-based biohybrids in the future biotechnological applications.

Cytochrome c oxidase (COX) represents the terminal enzyme complex of respiratory chain metabolic pathway and it occurs as monomer, dimer or as a part of respiratory supercomplexes in the inner mitochondrial membrane. COX assembly process is complicated, highly regulated and depends on many ancillary proteins. Mutations in COX subunits, which are encoded by mitochondrial and nuclear DNA, or in genes encoding its assembly proteins are frequent cause of very severe mitochondrial disorders. SURF1 assembly protein participates in the first steps of COX assembly, but its exact function is not yet clarified. In humans, mutations of SURF1 gene lead to severe COX defect and fatal neurodegenerative disorder, Leigh syndrome. Knockout of SURF1 gene in mouse causes isolated COX defect as well, but less pronounced and without involvement of CNS. The aim of the thesis was detailed analysis of disturbed COX biogenesis in a condition of SURF1 gene mutations or SURF1 gene knockout, from assembly of COX monomer to interaction of COX into supercomplexes, and to the impact of isolated COX defect on other OXPHOS complexes. Mutations of SURF1 gene in patient's fibroblasts led to marked accumulation of COX assembly intermediates and to a defect in formation of functional COX monomer, which was preferentially built into an...

Cytochrome c oxidase (COX) represents the terminal enzyme complex of respiratory chain metabolic pathway and it occurs as monomer, dimer or as a part of respiratory supercomplexes in the inner mitochondrial membrane. COX assembly process is complicated, highly regulated and depends on many ancillary proteins. Mutations in COX subunits, which are encoded by mitochondrial and nuclear DNA, or in genes encoding its assembly proteins are frequent cause of very severe mitochondrial disorders. SURF1 assembly protein participates in the first steps of COX assembly, but its exact function is not yet clarified. In humans, mutations of SURF1 gene lead to severe COX defect and fatal neurodegenerative disorder, Leigh syndrome. Knockout of SURF1 gene in mouse causes isolated COX defect as well, but less pronounced and without involvement of CNS. The aim of the thesis was detailed analysis of disturbed COX biogenesis in a condition of SURF1 gene mutations or SURF1 gene knockout, from assembly of COX monomer to interaction of COX into supercomplexes, and to the impact of isolated COX defect on other OXPHOS complexes. Mutations of SURF1 gene in patient's fibroblasts led to marked accumulation of COX assembly intermediates and to a defect in formation of functional COX monomer, which was preferentially built into an...

In this thesis, phylogenomic and comparative structural analyses of several widespread energy converting enzymes were performed. The focus was on the major subfamilies of the enzymes that process nucleoside triphosphates (ATP and GTP) and on some key enzymes of the electron transfer chains. First, we analyzed the P-loop GTPases, RadA/RecA recombinases, chaperone GroEL, branched-chain α-ketoacid dehydrogenase kinases, chaperone Hsc70, actins, and membrane pyrophosphatases. In the each inspected family we could identify (1) members which were potassium-dependent and/or contained K+ ions in the active site, and (2) potassium-independent enzymes with lysine or arginine residues as catalytic groups that occupy the positions of potassium ions in the homologous, K+-dependent enzymes. Based on the results of our analyses, we suggest that the appearance of the K+-binding sites could precede in evolution the recruitment of positively charged residues (lysine or arginine "fingers") with the latter providing more possibilities to control the enzyme reactions. Second, we have described the distinctive features of a phylogenetically separated subfamily of rotary membrane ATPases which we named N-ATPases. The N-ATPases have a specific operon organization with two additional subunits, absent in other rotary ATPases, and a complete set of Na+-binding ligands in the membrane c-subunits. We made a prediction, which was later confirmed, that these enzymes are capable of Na+ translocation across the membrane and may confer salt tolerance on marine prokaryotes. Third, phylogenomic analysis of the cytochrome bc complexes suggests that these enzyme complexes initially emerged within the bacteria and were then transferred to archaea via lateral gene transfer on several independent occasions. Our analysis indicates that the ancestral form of the cytochrome bc complex was a b6f-type complex; the fusion of the cytochrome b6 and the subunit IV to a "long" cytochrome b of the cytochrome bc1 complexes could have happened in different lineages independently. Fourth, our phylogenomic and comparative structural analyses of the cytochrome bc1 complex and of cytochrome c allowed us to trace how these enzymes became involved in triggering of apoptosis in Metazoa. We could trace the emergence of a specific cardiolipin-binding site within the cytochrome bc complex and the evolution of structural traits that account for the involvement of the cytochrome c as a trigger of apoptosis in vertebrates.

No description available.

Cytochrome c oxidase (COX) is one of two known Cu-containing enzymes in mitochondria. Delivery and insertion of copper into COX are very complex processes that require multiple steps and involve a large number of assisting factors. One of the involved components is Cox11p, a copper binding protein in the inner mitochondrial membrane that is conserved from prokaryotes to eukaryotes. Cox11p is essential for respiratory growth and implicated in the assembly of the CuB site located in subunit Cox1p of COX. In the thesis the topology of Cox11p was determined and evidence for its association with the mitochondrial translation machinery is provided. The interaction of Cox11p with mitoribosomes is mediated by its single evolutionary conserved transmembrane segment and appears to be indirect and mediated by another conserved membrane protein(s). A model is proposed in which the CuB site is co-translationally formed by a transient interaction between Cox11p and the nascent Cox1p in the mitochondrial intermembrane space. In addition the genetic and biochemical characterization of S. pombe Cox11p homologue was performed. Two versions of cox11+ gene are detected in a haploid S. pombe genome. Cells lacking either of the cox11+ copies remain respiratory competent, whereas deletion of both S. pombe cox11+ alleles appears to result in either spore lethality or in severe decrease of spores viability. Thus, both versions of SpCox11p are functional and important. In S. pombe Cox11p exists as a tandem with the mitoribosomal protein Rsm22p. This precursor protein is cleaved during mitochondrial import into two mature protein species corresponding to Rsm22p- and Cox11p-like moieties.

In this work, the identification of the promoter for c-cytochromes Cyt c Id-1 in Ideonella dechloratans has been investigated. Additionally, the study examined investigating whether gene expression is affected by the presence and absence of oxygen. This was investigated by amplifying the promoter region of Cyt c Id-1 (389 bp) and then cloning it into a reporter vector lacking a functional promoter for the upstream gene β-galactosidase. The reporter vector was transformed into E. coli RM101 and grown under both aerobic and anaerobic conditions. To examine the gene expression of Cyt c Id-1 the activity of β-galactosidase has been measured in both the aerobic and anaerobic cultures. The result showed that the gene is induced more in an anaerobic environment than in an aerobic environment, by comparing the activity of β-galactosidase under aerobic and anaerobic conditions. / I detta arbete har identifiering av promotorn för c-cytokromer Cyt c Id-1 i Ideonella dechloratans undersökts, samt att undersöka om genuttrycket påverkas av närvaro och frånvaro av syre. Detta är undersökts genom att amplifiera promotorregionen för Cyt c Id-1 (389 bp) för att sedan klona in den in i en reportervektor som saknar en fungerande promotor för uppströms genen β-galaktosidas. Reportervektorn transformerades till E. coli RM101 och odlades under både aeroba och anaeroba förhållanden.  För att undersöka genuttrycket av Cyt c Id-1 har aktiviteten hos β-galaktosidas uppmätts i både de aeroba och anaeroba odlingarna. Resultatet visat att genen induceras mer i anaerob miljö än i aerob miljö, genom att jämföra aktiviteten av β-galaktosidas under både aerob och anaerob förhållande.