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Supercomplexes of Prokaryotic Aerobic Respiratory Chains Escherichia Coli and Bacillus Subtilis Supramolecular Assemblies Pedro M.F

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Supercomplexes of Prokaryotic Aerobic Respiratory Chains Escherichia Coli and Bacillus Subtilis Supramolecular Assemblies Pedro M.F Supercomplexes of Prokaryotic Aerobic Respiratory Chains Escherichia coli and Bacillus subtilis supramolecular assemblies Pedro M.F. Sousa Dissertation presented to obtain the Ph.D degree in Biochemistry Instituto de Tecnologia Química e Biológica | Universidade Nova de Lisboa Instituto de Investigação Científica Tropical Oeiras, December, 2013 Supercomplexes of Prokaryotic Aerobic Respiratory Chains Escherichia coli and Bacillus subtilis supramolecular assemblies Pedro M.F. Sousa Dissertation presented to obtain the Ph.D degree in Biochemistry Instituto de Tecnologia Química e Biológica | Universidade Nova de Lisboa Instituto de Investigação Científica Tropical Supervisor: Ana M.P. Melo Co-supervisor: Miguel Teixeira Oeiras, December, 2013 From left to right: Prof. Miguel Teixeira, Dr. Lígia Saraiva, Prof. Carlos Romão, Prof. Thorsten Friedrich, Pedro Sousa, Dr. Margarida Duarte, Prof. João Arrabaça, Dr. Ana Melo. Instituto de Investigação Científica Tropical (IICT) Rua da Junqueira Nº86, 1º 1300-344 Lisboa Portugal Tel. (+351) 213 616 340 Instituto de Tecnologia Química e Biológica (ITQB) Av. da República Estação Agronómica Nacional 2780-157 Oeiras Portugal Tel. (+351) 214 469 323 The molecular cup is now empty. The time has come to replace the purely reductionist “eyes-down” molecular perspective with a new and genuinely holistic “eyes-up” view of the living world, one whose primary focus is on evolution, emergence, and biology’s innate complexity. Carl R.Woese (1928 – 2012) in “A new biology for a new century” Microbiology and Molecular Biology Reviews 68(2), 173 – 186 (2004) Acknowledgements The work summarized in this thesis is the product of several collaborations established during my research grant. It is with great appreciation that I would like to acknowledge the following groups and people: Ana Melo, my supervisor, for her endless support on my work. I’m truly honored of being her first PhD student and I have learned with Ana how to be constantly motivated, focused, and above all, how to critically analyze scientific results. I appreciate her confidence in my work, friendship and her ability to promote useful discussions. Miguel Teixeira, my co-supervisor, for his support on my work, useful discussions, and for the opportunity conceded. Lígia Saraiva and Célia Romão, for following the work developed these years and promoting fruitful discussions. Marco Videira, Filipe Santos, Sara Silva and Levi Bolacha, for the outstanding contribution they have provided to the work reported in this thesis. Furthermore, I would like to acknowledge Sara for supporting me all the times I needed, with unconditional love and friendship. For providing a friendly environment and a stimulating workplace, I would like to thank all my colleagues from IICT, namely, José Ramalho, Ana Ribeiro, António Leitão, Luís Goulão, Ana Fortunato, Paula Batista-Santos, Inês Graça, Elisabete Lopes, Paula Alves, Isabel Palos, Micaela Sousa and Tiago David. Brian Hood and Thomas Conrads from the Gynecologic Cancer Center of Excellence, Women’s Health Integrated Research Center at Inova Health System, USA and Nuno Charro, Fátima Vaz and Deborah Penque from the Proteomics Laboratory at Instituto Nacional de Saúde Dr Ricardo Jorge, for their contribution with the mass spectrometry sequencing of the supercomplexes described herein. Andreas Bohn and Luís Goulão from ITQB and IICT, respectively, for the valuable insights on the statistical analysis performed in this work. João Carita from ITQB is acknowledged for the large scale growth of Escherichia coli wild-type and mutant strains. Natalya Dudkina and Egbert Boekema from Groningen University, Netherlands, for valuable insights on the methodology used in this work regarding the electrophoretic analysis of supercomplexes. Julia Steuber and Thomas Vorburger from Stuttgart University, for allowing me to work on their laboratory and for their useful discussions. My family and friends, for providing me the knowledge and environment I needed to become a better person. I thank Luís Sousa, Maria Sousa, Ana Sousa, Sara Silva, Ricardo Alves, Mário Coelho and Bruno Pais. Fundação para a Ciência e Tecnologia (FCT) for my fellowship SFRH/BD/46553/2008. II Thesis publications Pedro M.F. Sousa, Sara T.N. Silva, Brian L. Hood, Nuno Charro, João N. Carita, Fátima Vaz, Deborah Penque, Thomas P. Conrads, Ana M.P. Melo (2011), “Supramolecular organizations in the aerobic respiratory chain of Escherichia coli”, Biochimie 93, 418-425. Pedro M.F. Sousa, Marco A.M. Videira, Andreas Bohn, Brian L. Hood, Thomas P. Conrads, Luis F. Goulao, Ana M.P. Melo (2012), “The aerobic respiratory chain of Escherichia coli: from genes to supercomplexes”, Microbiology 158, 2408-2418. Pedro M.F. Sousa, Marco A.M. Videira, Ana M.P. Melo (2013), “The formate:oxygen oxidoreductase supercomplex of Escherichia coli aerobic respiratory chain”, FEBS Lett. 587, 2559-2564. Pedro M.F. Sousa, Marco A.M. Videira, Filipe A.S. Santos, Brian L. Hood, Thomas P. Conrads, Ana M.P. Melo (2013), “The bc:caa3 supercomplexes from the gram positive bacterium Bacillus subtilis respiratory chain: a megacomplex organization?”, Arch. Biochem. Biophys. 537, 153-160. Other publications not included in this thesis Pedro M.F. Sousa, Marco A.M. Videira, Thomas Vorburger, Sara T.N. Silva, James W. Moir, Julia Steuber, Ana M.P. Melo (2012), “The novel NhaE-type Na+/H+ antiporter of the pathogenic bacterium Neisseria meningitidis” Arch. Microbiol. 195, 211-217. III IV Dissertation Abstract Aerobic respiratory chains are composed of a series of membrane complexes that catalyze the electron transfer from reducing substrates to oxygen. The energy released through this process is used to translocate protons across the membranes, thus generating a proton motive force that activates F1FO-ATP synthase to synthesize ATP. The arrangement of these enzymes in the inner mitochondrial membrane is well characterized in mammalian mitochondria, where different sets of supramolecular assemblies, or supercomplexes, involving the majority of the respiratory complexes were described, reinforcing the idea of an operational solid state model wherein the oxidative phosphorylation processes are optimized. A broader perspective of the ubiquity of such assemblies is obtained when analyzing the prokaryotic components that lead to oxygen utilization. The aerobic respiratory chain of these organisms is more flexible than the mitochondrial one, bearing alternative pathways in response to the metabolic needs of the cell, which reflect the ability of these organisms to cope with extreme environmental conditions. The PhD thesis here presented focuses on the identification and characterization of Escherichia coli and Bacillus subtilis aerobic respiratory chains supercomplexes, taking advantage of the milder solubilization properties of non- ionic detergents such as digitonin, which has allowed the isolation of such supramolecular assemblies in the respiratory chains of other organisms. Solubilized membrane components from wild type and respiratory chain mutant strains were analyzed by Blue Native Polyacrylamide Gel Electrophoresis (BN- PAGE) where heme staining and in gel enzymatic activities detection allowed the identification of protein complexes with higher molecular masses than those deduced for the individual complexes of both organisms respiratory chains. V The Escherichia coli aerobic respiratory chain is composed of at least six enzymes, namely type I (NDH-1) and II (NDH-2) NADH:quinone oxidoreductases, succinate:quinone oxidoreductase (SQR), cytochrome bo3 oxygen reductase and type I and II cytochromes bd oxygen reductases, that accomplish the transfer of electrons from the reducing substrates NADH and succinate to O2, with proton translocation through NDH-1 and cytochrome bo3 oxygen reductase. Other enzymes such as lactate, formate (FDH-O) and glycerol-1-phosphate dehydrogenases may also participate in this process. In this thesis, the supercomplexes of E. coli aerobic respiratory chain were reported for the first time. Prior to supercomplex detection, a thorough spectroscopic and kinetic characterization was performed in E. coli membranes, allowing the detection of the respiratory chain components operating in this Gram negative bacterium. The analysis of supercomplex composition in this bacterium revealed at least three distinct supercomplexes, one resulting from the association of NADH:quinone oxidoreductases NDH-1 and NDH-2, one composed of FDH-O and cytochromes bo3 and bdI oxygen reductases, and another comprising SQR and cytochrome bdII oxygen reductase. In addition, two homo-oligomers were described in this respiratory chain, namely the trimeric assembly of SQR and the dimeric form of cytochrome bo3 oxygen reductase. To establish the composition of NADH:ubiquinone oxidoreductase supercomplex three fundamental requirements were obtained, i) the detected BN-PAGE NADH:NBT oxidoreductase active band apparent mass of 606 ± 5 kDa is in agreement with the sum of NDH-1 and NDH-2 individual masses; ii) several peptides of NDH-1 subunits were identified by MALDI-TOF/TOF and LC-MS/MS analysis; and iii) the same band was undetected in solubilized membranes of E. coli strains devoid of NDH-1 or NDH-2 enzymes. Interestingly, NDH-1 stability seems to be influenced by the presence of NDH-2, since no free NDH-1 complexes were detected in the BN-PAGE analysis. VI Concerning the formate:oxygen oxidoreductase supercomplex (FdOx), the same requirements were obtained. Its corresponding BN-PAGE band, detected by NADH:
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