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Structural Characterisation of Nitrogen Fixation by the Enzyme Nitrogenase

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Structural Characterisation of Nitrogen Fixation by the Enzyme Nitrogenase I Structural characterisation of nitrogen fixation by the enzyme nitrogenase of Azotobacter vinelandii INAUGURALDISSERTATION zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Albert-Ludwigs-Universität Freiburg im Breisgau vorgelegt von Eva-Maria Michaela Burger aus Alzenau i. Ufr. 2015 II Vorsitzender des Promotionsausschusses: Prof. Dr. Stefan Weber Referent/in: Prof. Dr. Oliver Einsle Korreferent/in: Prof. Dr. Susana Andrade Datum der mündlichen Prüfung: 30.06.2015 III Indes sie forschten, röntgten, filmten, funkten, entstand von selbst die köstlichste Erfindung: der Umweg als die kürzeste Verbindung zwischen zwei Punkten. Erich Kästner Table of content 1. Zusammenfassung 1 2. Summary 3 3. Introduction 4 3.1 Nitrogen cycle 4 3.2 Biological nitrogen fixation 5 3.3 Nitrogenase 7 3.3.1 MoFe protein 7 3.3.1.1 FeMoco 8 3.3.1.2 P-cluster 12 3.3.2 Fe protein 14 3.3.3 Complex of MoFe and Fe protein 16 3.3.4 Mechanism 19 3.3.4.1 Fe protein cycle 20 3.3.4.2 MoFe cycle 20 3.3.5 Other substrates and inhibitors 23 3.4 Azotobacter vinelandii 26 3.4.1 Metabolism 26 2.4.2 Genome analysis of Azotobacter vinelandii 28 3.4.3 Assembly of MoFe protein in Azotobacter vinelandii 30 3.5 X-ray crystallography with proteins 33 3.5.1 Protein crystallography 33 3.5.2 X-ray diffraction 34 4. Material and methods 39 4.1 Microbiological methods 39 4.1.1 Bacterial strain 39 4.1.2 Media preparation 39 4.1.3 cultivation of Azotobacter vinelandii 40 4.1.4 Glycerol stock of Azotobacter vinelandii 42 4.1.5 Whole cells nitrogenase acetylene reduction assay (ARA) 42 4.2 Protein chemical methods 43 4.2.1 Anoxic techniques 43 I Table of content 4.2.2 Buffers and solutions 43 4.2.3 Preparation of MoFe protein and Fe protein 45 4.2.4 SDS-polyacrylamide-gel electrophoresis (SDS-PAGE) 47 4.2.5 Protein concentration determination (BCA-assay) 47 4.2.6 Electron paramagnetic resonance (EPR spectroscopy) 48 4.2.7 Crystallisation under anoxic conditions 49 4.2.7.1 Crystallisation of MoFe protein out of turnover with CO or acetylene 50 4.2.7.2 Crystallisation of MoFe and Fe protein in complex out of turnover with and without acetylene 52 4.2.8 Protein structure determination by molecular replacement 54 5. Results and discussion 55 5.1 Protein isolation of MoFe and Fe protein from A. vinelandii 55 5.2. Activity assay with pure protein 59 5.2.1 Crystallisation of MoFe protein after turnover with CO 61 5.2.2 Crystallisation of MoFe protein after turnover with acetylene 64 5.2.3 Discussion: Crystallisation of MoFe protein under CO and acetylene atmosphere 66 5.3 Crystallisation of MoFe and Fe protein in complex 74 5.3.1 Crystallisation of the complex out of turnover 74 5.3.2 crystallisation of the complex after acetylene turnover 78 5.3.3 Discussion: Crystallisation of the complex with and without acetylene 80 5.4 A model for the nitrogenase reaction 86 5.5 Outlook 91 6. References 93 II Zusammenfassung 1. Zusammenfassung Nitrogenase ist das einzig bekannte Enzymsystem, das in der Lage ist, elementaren Stickstoff in bioverfügbares Ammonium umzuwandeln. Das am besten untersuchte System ist die Mo-abhängige Nitrogenase aus Azotobacter vinelandii. Während der ATP-abhängigen Reaktion wird ein Komplex aus zwei Metalloproteinen, MoFe- und Fe-Protein, gebildet. Der Mechanismus der Fixierung ist nicht im Detail bekannt, da beispielsweise die Koordinationsstelle des Stickstoffs nicht klar ist. Die Strukturen der beiden beteiligten Proteine wurden mittels Röntgendiffraktometrie gelöst. Auch der Komplex aus beiden Proteinen wurde mit Hilfe von nicht hydrolysierbaren ATP-Analogen kristallisiert. Bei all diesen Versuchen wurden die beteiligten Proteine erst während der Kristallisation gemischt. Obwohl die Proteinstrukturen verfügbar sind, wurde bisher weder Substrat noch Inhibitor in den Strukturen gefunden. Ziel dieser Arbeit war die Kristallisation des MoFe-Proteins aus einem non- resting state heraus, um Substrate in der Struktur nachweisen zu können. Dafür wurde der etablierte Aktivitätsnachweis für Nitrogenase als Grundlage verwendet. Der Nachweis basiert auf der Reduktion von Acetylen zu Ethylen durch MoFe-Protein in Gegenwart von Fe-Protein und ATP. Der Ansatz wurde den Kristallisationsbedingungen angepasst und mit CO als Inhibitor und Acetylen als Substrat durchgeführt. Zusätzlich wurde der Komplex aus MoFe- und Fe-Protein aus der Reaktion heraus kristallisiert. Dafür wurde das ATP- Analoge AMPPCP verwendet. Dieser Versuch wurde auch in Gegenwart von Acetylen durchgeführt. Alle erhaltenen Strukturen wurden mit publizierten Strukturen verglichen, wobei der Fokus auf dem aktiven Zentrum, dem FeMoco, lag. Für eine besseres Verständnis der elektronischen Situation am FeMoco, wurden sog. slice maps mit dem Programm PyMOL erzeugt. Es konnte gezeigt werden, dass CO die Bindesituation im Cluster verändert und ein überbrückendes Schwefelatom 1 Zusammenfassung ersetzt. Im Fall von Acetylen als Substrat kommt es wohl ebenfalls zu einer Veränderung in der elektronischen Situation am FeMoco. 2 Summary 2. Summary Nitrogenase is the only known enzyme that is able to fix elemental nitrogen to bioavailable ammonia. The best understood system is the Mo dependent nitrogenase of Azotobacter vinelandii. During the ATP dependent reaction, a complex of two metal proteins, the MoFe protein and the Fe protein, is formed. The mechanism of the fixation is not known in detail, as for example the binding site of the substrate N2 is still under debate. The protein structure of both involved proteins was solved by X-ray diffraction. Additionally, the complex of both proteins was crystallised by the usage of ATP-analogues. In all experiments, the proteins were directly mixed during the crystallisation process. Although the crystal structures are available, it was not possible so far to see any substrate or inhibitor inside the structure. This work focuses on the crystallisation of the MoFe protein out of a non- resting state in order to get any substrate into the protein. To this end, the well-established activity assay of the nitrogenase system was used. This assay is based on the reduction of acetylene to ethylene by the MoFe protein in presence of the Fe protein and ATP. The assay was adjusted to crystallisation conditions and carried out with CO as inhibitor or acetylene as alternative substrate. In addition, the crystallisation of the complex of MoFe and Fe protein out of the turnover state was done. For that, the ATP-analogue AMPPNP was used. This experiment was also done with addition of acetylene. All obtained structures were compared to published structures. The focus was there on the situation around the active site, the FeMoco. For a better understanding the electron situation was visualized with map slices in the program PyMOL. It could be shown that for CO the binding inside the cluster changes, as one of the bridging sulphurs of the cluster is replaced by the CO. For acetylene as substrate a change in the electronic situation at the FeMoco is indicated. 3 Introduction 3. Introduction 3.1. Nitrogen cycle More than 99% of the free nitrogen on Earth are found in the air, where dinitrogen gas is the main component (78.09 Vol.-%) before oxygen (20.95 Vol.- %). The main form of bound nitrogen is nitrate, for example NaNO3 (Chile saltpetre). Nitrogen is an essential compound of all biopolymers, like proteins and nucleic acids, in all organisms on earth. Nitrogen in this form of NH3 can also be found in the atmosphere of different planets, together with methane, and is therefore believed to be an important substance for the development of a biosphere of the Earth. (Holleman & Wiberg, 1985) Although roughly 80% of air consist of dinitrogen gas, the bioavailability of nitrogen is quite limited. The reason for this is the extraordinary chemical stability of the triple bond of the N2-molecule. For dissociation, an energy of 946 kJ mol-1 is needed. (Holleman & Wiberg, 1985) Only a small number of unicellular organisms, like some archaea or bacteria, are able to fix dinitrogen out of air and convert it into ammonia, which is then bioavailable for other organisms, especially for plants to generate biomass (Einsle and Kroneck, 2004). The transformation of nitrogen to ammonia is also possible in a technical chemical reaction. Fritz Haber invented the process in the beginning of the 20th century. For his work he was honoured with the Nobel Prize in 1918. The process was further improved by Carl Bosch (Nobel Prize 1931) for the industrial production of ammonia. Until today the Haber-Bosch-process (reaction 1) produces 90% of all ammonia worldwide. (Holleman & Wieberg, 1985) 3H2 + N2 2NH3 + 92.28 kJ (1) 4 Introduction The reaction needs a lot of energy in form of high temperature (500 °C), high pressure (200 bar) and a catalyst. The yield of ammonia is then 17.6 Vol.-%. The catalyst consists of Fe3O4 and Fe2O3 with small amounts of Al2O3, K2O and CaO. The reactive part is the α-Fe, the other components work like a promoter for the reaction (Holleman & Wieberg, 1985). 3.2 Biological nitrogen fixation Nitrogen is able to exist in different redox states from –III to +V. Enzymes convert the change between the different states. The relationships between the reactions are shown in figure 1.1 (Einsle & Kroneck, 2004). 2 3 4 5 6 1 Fig. 3.1: Nitrogen cycle in biology. The oxidation state of the different nitrogen compounds is shown on the left side. The involved proteins are shown for every process (1) – (6). (1) Nitrogen fixation, (2) denitrification, (3) nitrification, (4) and (5) assimilatory and dissimilatory nitrate ammonification and (6) anaerobic ammonia oxidation (annamox) (Einsle & Kroneck, 2004). In the 19th century, the phenomenon of nitrogen fixation by legumes was described by Hermann Hellriegel and Hermann Wilfarth (Wilfarth & Hellriege, 5 Introduction 1888).
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