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Studies on Catalytic Mechanism of [Fe]- Hydrogenase from Methanogenic Archaea Based on Crystal Structures

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Studies on Catalytic Mechanism of [Fe]- Hydrogenase from Methanogenic Archaea Based on Crystal Structures Studies on catalytic mechanism of [Fe]- hydrogenase from methanogenic archaea based on crystal structures DISSERTATION zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Biologie der Philipps-Universität Marburg Vorgelegt von Gangfeng Huang aus Shaoxing, China Marburg/Lahn, Deutschland, 2019 Die Untersuchungen zur vorliegenden Arbeit wurden in der Zeit von September 2015 bis Juni 2019 am Max-Planck-Institut für terrestrische Mikrobiologie in Marburg/Lahn unter der Leitung von Dr. Seigo Shima durchgeführt. Vom Fachbereich Biologie der Philipps-Universität in Marburg/Lahn als Dissertation angenommen am: Erstgutachter: Dr. Seigo Shima Zweitgutachter: Prof. Dr. Johann Heider Tag der mündlichen Prüfung: ERKLÄRUNG Hiermit versichere ich, dass ich meine Dissertation mit dem Titel "Studies on catalytic mechanism of [Fe]-hydrogenase from methanogenic archaea based on crystal structures " selbständig und ohne unerlaubte Unterstützung angefertigt und mich dabei keiner anderen als der von mir ausdrücklich bezeichneten Quellen und Hilfen bedient habe. Die Dissertation wurde weder in der jetzigen noch in einer ähnlichen Form bei einer anderen Hochschule eingereicht und hat keinen sonstigen Prüfungszwecken gedient. Marburg, den 03.2019 Gangfeng Huang Publications Part of this dissertation was published as below: 1. Huang G, Wagner T, Ermler U, Bill E, Ataka K, Shima S. Dioxygen sensitivity of [Fe]-hydrogenase in the presence of reducing substrates. Angew. Chem. Int. Ed. 2018, 57: 4917-4920. 2. Wagner T*, Huang G*, Ermler U, Shima S. How [Fe]-hydrogenase from Methanothermobacter is protected against light and oxidative stress. Angew. Chem. Int. Ed. 2018, 57: 15056-15059. 3. Huang G*, Wagner T*, Wodrich M, Ataka K, Bill E, Ermler U, Hu X, Shima S. The atomic-resolution crystal structure of activated [Fe]-hydrogenase. (A research article in revision.) Other Publications: 4. Bai L, Fujishiro T, Huang G, Koch J, Takabayashi A, Yokono M, Tanaka A, Xu T, Hu X, Ermler U, Shima S. Towards artificial methanogenesis: biosynthesis of the [Fe]-hydrogenase cofactor and characterization of the semi-synthetic hydrogenase. Faraday Discussion 2017, 198, 37-58. 5. Watanabe T, Wagner T, Huang G, Kahnt J, Ataka K, Ermler U, Shima S. The bacterial [Fe]-hydrogenase paralog uses tetrahydrofolate derivatives as substrates. Angew. Chem. Int. Ed. 2019, 58: 3506-3510. 6. Pan H, Huang G, Wodrich M, Tirani F, Ataka K, Shima S, Hu X. A catalytically active [Mn]-hydrogenase incorporating a non-native metal cofactor. Nature Chemistry 2019, In press. 7. Huang G*, Wagner T*, Ermler U, Shima S. Multiple conformational changes involved in the catalytic cycle of methylene-tetrahydromethanopterin dehydrogenase (MtdA) (in preparation) * These authors contributed equally to these works. “To ordain conscience for Heaven and Earth, to secure life and fortune for the people, to continue lost teachings for past sages, to establish peace for all future generations.” Zai Zhang (1020–1077), philosopher and cosmologist Contents Abbreviations ...............................................................................................................1 Summary .....................................................................................................................3 Zusammenfassung ......................................................................................................4 1. Introduction ..............................................................................................................6 1.1 Hydrogenases .......................................................................................................6 1.2 [Fe]-hydrogenase in methanogens ...................................................................... 10 1.3 Current catalytic mechanism studies of [Fe]-hydrogenase .................................. 15 1.4 Aim of this study .................................................................................................. 17 2. Results .................................................................................................................. 18 2.1 Insights into catalytic mechanism of [Fe]-hydrogenase ....................................... 18 2.1.1 Crystal structures of [Fe]-hydrogenase in the open and closed forms .............. 18 2.2.2 Mössbauer and IR spectroscopic analysis ........................................................ 26 + 2.2.3 Structures of the FeGP cofactor and methenyl-H4MPT ................................... 29 2.1.4 A proposed catalytic mechanism of [Fe]-hydrogenase ..................................... 31 2.2 Evidence for the presence of iron-hydride as the catalytic intermediate .............. 35 2.2.1 [Fe]-hydrogenase catalyzes reduction of O2 to H2O2 ........................................ 35 2.2.2 The crystal structure of O2-inactivated [Fe]-hydrogenase ................................. 43 2.2.3 Spectroscopic analyses of O2-inactivated [Fe]-hydrogenase ............................ 46 2.2.4 The proposed iron-hydride intermediate for O2 reduction to H2O2 .................... 49 2.3 Protection-regulation mechanism of hexameric [Fe]-hydrogenase ...................... 51 2.3.1 The crystal structure of [Fe]-hydrogenase (M. marburgensis) in hexamer ....... 51 2.3.2 Dissociation of Hexameric assembly ................................................................ 54 2.3.3 Hexameric [Fe]-hydrogenase is resistant to light and oxidative stresses ......... 56 2.3.4 The strategy of [Fe]-hydrogenase against light and oxidative stresses ............ 58 3. Discussion ............................................................................................................. 60 4. Materials and methods .......................................................................................... 63 4.1 Materials .............................................................................................................. 63 4.1.1 Chemicals ......................................................................................................... 63 4.1.2 Strains .............................................................................................................. 63 4.1.3 Media ................................................................................................................ 63 4.2 Methods ............................................................................................................... 63 4.2.1 Cultivation of M. marburgensis ......................................................................... 63 4.2.2 Genes synthesis of [Fe]-hydrogenase from M. aeolicus ................................... 64 4.2.3 Heterologously production of the apoenzyme of [Fe]-hydrogenase .................. 65 4.2.4 Purification of the apoenzyme of [Fe]-hydrogenase ......................................... 65 4.2.5 Purification of [Fe]-hydrogenase from M. marburgensis ................................... 66 4.2.6 Extraction of FeGP cofactor from [Fe]-hydrogenase ........................................ 66 4.2.7 Reconstitution of [Fe]-hydrogenase holoenzyme in vitro .................................. 67 4.2.8 [Fe]-hydrogenase enzyme activity assay .......................................................... 67 4.2.9 Crystallization ................................................................................................... 67 4.2.10 Structure analysis ........................................................................................... 69 4.2.11 Infrared spectroscopy ..................................................................................... 71 4.2.12 Mössbauer spectroscopy ................................................................................ 72 4.2.13 QM/MM computations..................................................................................... 73 4.2.14 H2O2 measurement ......................................................................................... 73 4.2.15 O2 measurement .......................................................................................... 74 4.2.16 UV-Vis spectroscopy ...................................................................................... 75 4.2.17 Electron paramagnetic resonance spectroscopy ............................................ 75 4.2.18 Size-exclusion chromatography ...................................................................... 75 4.2.19 Ultrafiltration to determine the oligomerization state of [Fe]-hydrogenase ...... 76 4.2.20 Light-inactivation of [Fe]-hydrogenase ............................................................ 76 4.2.21 Inactivation of [Fe]-hydrogenase by oxidative stress ...................................... 76 References ................................................................................................................ 77 Supplementary information ........................................................................................ 85 Acknowledgements ................................................................................................... 95 Curriculum vitae ......................................................................................................... 98 1 Abbreviations Abbreviations Hmd H2-forming methylene-tetrahydromethanopterin dehydrogenase ([Fe]-hydrogenase) mHmd [Fe]-hydrogenase from Methanothermobacter marburgensis jHmd [Fe]-hydrogenase from Methanocaldococcus jannaschii aHmd [Fe]-hydrogenase from Methanococcus aeolicus F420 Coenzyme F420 Frh F420-reducing [NiFe]-hydrogenase Mtd F420-dependent methylenetetrahydromethanopterin
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