Open Access

Priya Gurumoorthy

• In mitochondria, biogenesis of oxidase is a crucial process involving the participation of an array of assembly factors. Studying the process of biogenesis in eukaryotes is highly complicated due to the presence and partaking of two genetic systems. Employing a bacterial model such as Paracoccus denitrificans that utilizes only one genetic system enables easy studying of the assembly process. The aa3 cytochrome c oxidase of P. denitrificans shows high structural and functional homology to its mitochondrial counterpart despite its simple subunit composition. The assembly of the core subunits I and II that house the active redox centers (heme a, and heme a3.CuB centre in subunit I; and the binuclear CuA centre in subunit II) along with the chaperons responsibly for their incorporation form the crux of this work. This work concentrates particularly on CtaG, a chaperone previously speculated to be involved in the delivery of copper to the CuB center in subunit I. As the full length structure of CtaG or its structural homologues have not been solved, attempts were made to obtain high-diffracting crystals of CtaG by heterologously expressing it in E. coli. Growth media, expression strains and induction parameters were some of the conditions screened in order to obtain optimal yield. Additives, pH and detergent were screened to yield a homogeneous preparation of CtaG. Crystallization trials were conducted by employing the sitting drop, vapour diffusion, method and later the bicelles were employed. Preliminary crystals obtained were further optimized employing seeding, detergent and additives, to improve diffraction. The diffraction improved from 30 Å to 15 Å. BN PAGE (Blue Native Polyacrylamide Gel Electrophoresis) analysis and cross-linking studies were undertaken to decipher the oligomeric condition of CtaG. Both the methods indicate that the protein is a dimer under native conditions. To study the importance of CtaG in the process of oxidase assembly, two deletion mutants were obtained from the lab; one with only ctaG deleted and the other with ctaG and most of the upstream ORF. The effect of the deletion was assayed on the assembly and activity of oxidase. The deletion mutants showed residual activity of approx. 20 %, while displaying a very low heme signal (both in membranes and in purified COX). In order to exclude polar effects arising due to gene manipulation, complementation strains were prepared, reintroducing ctaG alone into both the deletion strains. Complementation strains, where only ctaG was deleted and re-introduced assayed for COX activity showed a restoration in activity to approx. 70 %. Further, calculating the heme:protein ratio, the deletion strains displayed a value of 7 nmol/mg of oxidase which was increased to wild type levels of 16 nmol/mg in the complementation strains. To further confirm the absence of the copper in subunit I, total reflection X-ray fluorescence spectroscopy analysis was carried out, which showed a decrease in the copper content in the deletion strain, restored on complementation. The strain lacking in the ORF and ctaG when complemented with ctaG alone illustrated no increase in activity or heme signal in comparison to that of the deletion strain. These point at a possible role for ORF in the assembly of COX, which is still absent in the complementation strains. To further characterize the ORF, a series of bioinformatical analysis was carried out, the results from which were insufficient to characterize the ORF conclusively. In order to enlist the proteins involved in the biosynthesis of COX, two independent approaches were employed. Two-dimensional gel examinations of solubilised membranes from untreated and cross-linked cells were analyzed by Western blotting. The CtaG-COX interaction was observed in untreated membranes, which was additionally strengthened by cross-linking. To further confirm this association, pull-down assays were done employing protein A coated magnetic beads coated with different antibodies and incubated with solubilised membranes derived from untreated or cross-linked cells. The elutions were assayed by Western blotting and confirmed for the CtaG-COX interaction. These fractions were further analysed by mass spectrometry to identify other chaperons involved in biogenesis of oxidase. Along with CtaG, I also noticed Sco, Surf1c and other factors involved in the recruitment and transport of heme (CtaB, CtaA, and Ccm proteins). Interestingly, protein components of both ribosomal subunits and protein translocation factors were observed, which indicated a co-translational approach for co-factor insertion into COX.
• Die Cytochrom c Oxidase bildet den terminalen Komplex der Elektronentransportkette in Mitochondrien und vielen Bakterien. Sie spielt eine zentrale Rolle bei der Reduktion von molekularem Sauerstoff zu Wasser. Die Elektronen werden sequentiell von Cytochrom c auf die Redoxzentren der Oxidase übertragen. Das CuA-Zentrum in Untereinheit II ist der primäre Elektronenakzeptor, der die Elektronen anschließed an das Häm a- und dann an das Häm a3·CuB-Zentrum (binukleares Zentrum) in Untereinheit I weiterleitet, wo auch die Sauerstoffreduktion stattfindet. Die Biogenese der mitochondrialen Oxidase is ein streng regulierter Prozess, der auf das Zusammenspiel einer Vielzahl von Assemblierungsfaktoren angewiesen ist. Fehler im Assemblierunsprozess führen zu fatalen neurologischen Erkrankungen wie dem Leigh Syndrom. Die Untersuchung der Biogenesevorgänge in Eukaryonten ist hoch komplex, da zwei genetische Systeme vorhanden sind und aufeinander abgestimmt werden müssen. Die Verwendung eines bakteriellen Modellsystems wie Paracoccus denitrificans, das nur ein genetisches System besitzt, erleichtert die Untersuchung solcher Mechansimen. Die Häm-aa3-Cytochrom c Oxidase aus P. denitrificans zeigt trotz ihrer einfachen Untereinheiten-Zusammensetzung eine hohe strukturelle und funktionelle Homologie zu ihrem mitochondrialem Pendant. Die Biogenese der Kernuntereinheiten I und II, welche die aktiven Redoxzentren beherbergen, sowie die für ihren Einbau verantwortlichen Chaperone stehen im Mittelpunkt dieser Arbeit. Drei membrangebundene Chaperone sind in den Einbau der Redox-Cofaktoren in die Oxidase involivert: (i) Surf1c überträgt beide Häm-Moleküle auf Untereinheit I; (ii) Sco beliefert wahrscheinlich das CuA-Zentrum in Untereinheit II mit Kupfer; und (iii) CtaG baut vermutlich Kupfer im binuklearen Zentrum von Untereinheit I ein. Diese Arbeit beschäftigt sich ausschließlich mit dem Kupfer-Carrier CtaG, wobei der Fokus auf dessen Rolle und Bedeutung im Biogeneseprozess der Oxidase liegt. Drei unabhängige Strategien wurden herangezogen, um dessen essentielle Rolle im Assemblierunsprozess aufzuzeigen: strukturelle Charakterisierung, funktionelle Studien des Proteins und seine direkte Beteiligung an der Oxidase-Biogenese...