Beschreibung:
Abstract: In cells, oxygen is mainly consumed by integral membrane proteins, the oxidases. The use of oxygen as terminal electron acceptor of the respiratory chains enables organisms the generation of a proton motive force driven by a high-energy redox reaction. Among the enzymes catalyzing this reaction a major player is ubiquinol:oxygen cytochrome bd oxidoreductase, bd oxidase, a terminal procaryotic oxidase in the respiratory chains. Cytochrome bd oxidase is also found in many pathogens such as Salmonella enterica and Mycobacterium tuberculosis. The enzyme is not related to the oxidases of the heme-copper family. Cytochrome bd oxidase catalyzes the reduction of oxygen to water with simultaneous oxidation of ubiquinol to ubiquinone without producing reactive oxygen species. The exergonic redox reaction is coupled to the generation of a vectorial electrochemical proton gradient. The oxidase has a very high affinity towards oxygen and is indispensable for pathogenic organisms during host infection, which makes the enzyme interesting for antimicrobial drug development. For this purpose, detailed knowledge of the reaction mechanism and the enzyme structur is essential.<br>In the first part of this work the growth conditions at which Escherichia coli overproduces cytochrome bd oxidase were optimized. Different detergents for solubillization and purification of the membrane protein were tested. A novel preparation protocol was established resulting in large amounts of pure oxidase. By different crystallization experiments, conditions were found leading to a reproducible crystallization of bd oxidase. The crystals were measured at the Swiss Light Source at the Paul Scherer Institute in Villigen, Switzerland, without obtaining high-resolution diffraction data. Accordingly, an attempt was made to bring the native oxidase in a detergent-free state for cryo-electron microscopy (EM) experiments. The stabilizing detergent was replaced in several steps by an amphiphatic surfactant, amphipol A8-35. The bd oxidase in amphipols was measured in collaboration with Dr. Tim Rassmussen and Prof. Dr. Bettina Böttcher at the Rudolf-Virchow-Center of the Julius-Maximilians-University of Würzburg, Germany on a Titan Krios G3 equipped with a Falcon III detector. The structure of E. coli bd oxidase was solved at 3.3 Å resolution by cryo-EM. The structure of E. coli oxidase is highly similar to that of Geobacillus thermodenitrificans. However, there are small, but significant differences leading to a different mechanism of both homologous enzymes. A new subunit, CydY was identified, that blocks the oxygen channel on CydA, which is associated with the rearrangement of the heme b595 and d cofactors. The arrangement of the two heme groups in the E. coli enzyme was demonstrated by UV/vis redox difference spectrua of various mutants by a close examination of the density maps. The heme groups, in particular the chlorine-type heme d, did<br>not fit into the corresponding position of heme d in the G. thermodenitrificans bd oxidase. A ubiquinone-8 is bound to the subunit CydB, stabilizing the four subunit complex.<br>The family of cytochrome bd oxidase is divided into two subfamilies that are distinguished by the length of the so called Q-loop in CydA. Members of the S-subfamily containing a short Q-loop while members of the L-subfamily comprise a C-terminal extension. Variants of the E.coli enzyme from the L-subfamily with a shortened Q-loop were created, as well as an chimera, that carried the short Q-loop of G. thermodenitrificans bd oxidase. Removal of parts or of the entire Q-loop of E. coli bd oxidase led to a disturbed assembly or a rapid degradation of the enzyme in the membrane. The chimera of the E. coli bd oxidase containing the G. thermodenitrificans Q-loop was detected in the membrane, but could not be purified due to its instability. These results support the suggestion that the C-terminal extension of the Q-loop in E. coli bd is stabilizing the structure of the enzyme