Sulfur Oxygenase Reductases - A Structural and Biochemical Perspective vom Fachbereich Biologie der Technischen Universität Darmstadt zur Erlangung des akademischen Grades eines Doctor rerum naturalium genehmigte Dissertation vorgelegt von Dipl. Biol. Andreas Veith aus Seeheim-Jugenheim 1. Referent: Dr. habil. Arnulf Kletzin 2. Referent: Prof. Dr. Felicitas Pfeifer Eingereicht am 21.07.2011 Tag der mündlichen Prüfung: 16.09.2011 Darmstadt 2011 D 17 "Trying to determine the structure of a protein by UV spectroscopy was like trying to determine the structure of a piano by listening to the sound it made while being dropped down a flight of stairs." -- Francis Crick Die vorliegende Arbeit wurde als Promotionsarbeit am Institut für Mikrobiologie und Genetik des Fachbereichs Biologie der Technischen Universität Darmstadt unter Leitung von Herrn Dr. Arnulf Kletzin in der Abteilung von Frau Prof. Felicitas Pfeifer im Zeitraum von Mai 2007 bis Juli 2011 angefertigt. Ein Teil der vorliegenden Arbeit wurde in Portugal am Instituto de Tecnologia Químicia e Biológica, Universidade Nova de Lisboa (ITQB/UNL), Oeiras, Portugal in der Arbeitsgruppe von Dr. Carlos Frazão, Dr. Cláudio M. Gomes und Prof. Miguel Teixeira im Rahmen einer Kooperation durchgeführt. Ehrenwörtliche Erklärung Ich erkläre hiermit ehrenwörtlich, dass ich die vorliegende Arbeit selbstständig angefertigt habe. Sämtliche aus fremden Quellen direkt oder indirekt übernommenen Gedanken sind als solche kenntlich gemacht. Die Arbeit wurde bisher keiner anderen Prüfungsbehörde vorgelegt und noch nicht veröffentlicht. Darmstadt, den 21.07.2011 Andreas Veith Acknowledgements First and foremost I would like to thank my supervisor Dr. Arnulf Kletzin for giving me the opportunity to step into the fascinating world of proteins. I was in the lucky position to receive great support, encouragement and to find a door that was always open for questions and discussions. Special thanks go to Prof. Felicitas Pfeifer for taking the position of the co-advisor and for her extensive support throughout my work. And many thanks for making Darmstadt an outpost for the intriguing Archaea. I would like to express my deepest gratitude for Dr. Carlos Frazão (ITQB, Oeiras, Portugal), my supervisor of the crystallographic part of the thesis. He never got tired to teach crystallography to a raw beginner and to discuss results over and over again, even beyond my visits at ITQB. Putting much work into all SOR projects, he managed it to determine the HnSOR structure. Thanks to all members of the Molecular Xtallography group at ITQB, especially Catarina, Joana, Ana, Mario, José, Miguel, Matteo, Bruno and Przemek for their help and for sharing various wild-pig or Fado experiences. You made me feel extremely comfortable from the first day on! Specials thanks go to Tiago Bandeiras for his great passion for science (and Benfica) and many lunch-time discussions (Zé Varunca). I also would like to thank Ricardo Coelho for having a helping hand in growing and fishing crystals and also for taking care of the HnSOR, when I was absent. I would like to thank Prof. Miguel Teixeira (ITQB) for providing the EPR data. Dr. Cláudio M. Gomes (ITQB) and his entire group are gratefully acknowledged for the time at their lab. Special thanks go to Hugo Botelho for his great help during my short stay. Claudia Rittmeyer (Goethe-Universität, Frankfurt, Germany) is acknowledged for performing the TXRF measurements. Ok, this might take a while, but it cannot be missed out: a huge thank you goes to all former and present colleagues from the Kletzin & Pfeifer Lab, especially Ina Schmidt, Larissa Marschaus, Stella Tavlaridou, Regina Frommherz, Dennis Petrasch, Michael Forth, Matti Eckert, ―Cpt.‖ Fabian Müller, Bastian Naß and Gerald Losensky. Jonas Protze is acknowledged for providing mutant and biochemical data during his ―Forschungspraktikum‖ and for keeping the ÄKTA running. Katrin Faist and Gaby Liebing (who will do the ―back-scratching‖ now?) are especially acknowledged for their excellent assistance. Special thanks go to Renate Fröhlich, who shared the lab with me the past few years and always guaranteed a ―fresh‖ start to the day. Thanks also to Nam-Suk Polin, Cordula Bernhardt and Giuseppe Vetrano for making work a bit easier. Thanks to all of you for providing such an amazing working atmosphere and for sharing those great experiences in- and outside the lab. I would also like to thank all present and former colleagues from the third floor (AG Heider, AG Simon and AG Göringer). Tamara Heß is especially acknowledged for her great help all these years. Thanks go to Tim Urich for always finding time to discuss some new facts about the ―tiny and lovely‖ SOR. Mark Günzel and Till Albrecht are gratefully acknowledged for the correction of this work. Thanks to Andreas ―Flash‖ Klingl for the great ―heavy metal‖ interaction. The Mühlhiasl prophesized it! A very special thank you goes to Dr. Sabrina Fröls for personal care, encouragement, discussions and unlimited helpfulness throughout practical courses, diploma and PhD thesis. Jens Riede is thankfully acknowledged for his friendship and for his help to think outside the ―microbiology box‖. Thanks to Gabi, Thomas, Heinz, Anni and Kurt for their continuous support all these years. This would have never been possible without you!!! And more than thousand thanks to Nadine for her special support and her surpassing patience especially at the end of this work. Publications Parts of the present work and of other projects during the time of this thesis have already been published or have been submitted for publication. Veith, A., Klingl, A., Zolghadr, B., Lauber, K., Mentele, R., Lottspeich, F., Rachel, R., Albers, S. V. and Kletzin, A. (2009) Acidianus, Sulfolobus and Metallosphaera surface layers: structure, composition and gene expression. Mol Microbiol 73: 58-72. Botelho, H. M., Leal, S. S., Veith, A., Prosinecki, V., Bauer, C., Fröhlich, R., Kletzin, A. and Gomes, C. M. (2010) Role of a novel disulfide bridge within the all-beta fold of soluble Rieske proteins. J Biol Inorg Chem 15: 271-281. 1Veith, A., Urich, T., Seyfarth, K., Protze, J., Frazão, C. and Kletzin, A. (2011) Substrate pathways and mechanisms of inhibition in the sulfur oxygenase reductase of Acidianus ambivalens. Front Microbio 2:37. doi: 0.3389/fmicb.2011.00037 2Veith, A., Botelho, H. M., Gomes, C. M. and Kletzin, A. (2011) The Sulfur Oxygenase Reductase from the Mesophilic Bacterium Halothiobacillus neapolitanus is a Highly Active Thermozyme. Submitted June 2011 Veith, A., Coelho, R., Kletzin, A. and Frazão, C. (2011) Cloning, expression, purification, crystallization and preliminary crystallographic studies of the sulfur oxygenase reductase of Halothiobacillus neapolitanus DSM 15147. In preparation 1 The tetramer channel mutants Del L, Del K, F141A, F133A and F133A/F141A were originally constructed by Kerstin Seyfarth. The active site pore mutants M296V, M297A, MM296/297TT and MM296/297VT were constructed by Jonas Protze under my supervision in the course of this work. Mutants of the trimer channel and of the zinc-binding site were generated in the course of this work. The structural datasets of AaSOR complexed with iodoacetamide, zinc acetate or p-chloromercuribenzoic acid were initially obtained by Tim Urich and refined by Carlos Frazão (ITQB). 2All Far UV / CD spectroscopy experiments were performed under the guidance of Hugo Botelho (ITQB) in the group of Cláudio M. Gomes (ITQB) in the course of this work. All other experiments were carried out in Darmstadt in the course of this work. Index Summary ................................................................................................................................ 1 1. Introduction ................................................................................................................... 3 1.1. Elemental sulfur ..................................................................................................... 3 1.2. Microbial sulfur oxidation ..................................................................................... 4 1.2.1. Bacterial sulfur oxidation pathways ................................................................ 4 1.2.2. Archaeal sulfur oxidation pathways ................................................................ 7 1.2.3. Sulfur oxidation pathway of Acidianus ambivalens ........................................ 8 1.3. The sulfur oxygenase reductase ........................................................................... 10 1.4. Goals of this study ............................................................................................... 18 2. Materials and Methods ................................................................................................ 20 2.1. Materials .............................................................................................................. 20 2.1.1. Chemicals ...................................................................................................... 20 2.1.2. Microorganisms ............................................................................................. 21 2.1.3. Plasmids ......................................................................................................... 21 2.1.4. Enzymes and kits ........................................................................................... 21 2.1.5. Synthetic oligonucleotides ............................................................................. 22 2.1.6. Molecular weight standards