Anaerobic carbon monoxide dehydrogenase [Elektronische Ressource] : mechanism of CO-oxidation at the [NiFe_1tn4S_1tn4OH_1tnx] cluster and nickel-processing by its ATPase CooC / vorgelegt von Jae-Hun Jeoung

Jeoung

Sujets

• Biologie

Informations

• Publié par: universitat_bayreuth
• Publié le: 01 janvier 2008
• Nombre de lectures: 44
• Langue: Deutsch
• Poids de l'ouvrage: 85 Mo

Extrait

Anaerobic Carbon Monoxide Dehydrogenase:
Mechanism of CO-Oxidation at the [NiFe S OH ] Cluster 4 4 x
and Nickel-Processing by its ATPase CooC








DISSERTATION

zur Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)




der Fakultät für
Biologie, Chemie und Geowissenschaften
der Universität Bayreuth
vorgelegt von





Jae-Hun Jeoung
aus Korea






Bayreuth 2008



















Die Untersuchungen zur vorliegenden Arbeit wurden von April 2004 bis September
2008 an der Universität Bayreuth unter der Leitung von Herrn Prof. Dr. Holger Dobbek
durchgeführt.
The investigations of the present work were accomplished from April 2004 until
September 2008 at the University of Bayreuth under the supervision of Prof. Dr. Holger
Dobbek.










Vollständiger Abdruck der von der Fakultät für Biologie, Chemie und
Geowissenschaften der Universität Bayreuth genehmigten Dissertation zur Erlangung
des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.).




Promotionsgesuch eingereicht am: 15.10.2008
Tag des wissenschaftlichen Kolloquiums: 08.12.2008




Erster Gutachter: Prof. Dr. Holger Dobbek
Zweiter Gutachter: Prof. Dr. Matthias Ullmann
Vorsitzender: Prof. Dr. Andreas Fery
Prof. Dr. Stephan Clemens





Teile der im zeitlichen Rahmen dieser Dissertation erzielten Ergebnisse sind in
folgenden Publikationen veröffentlicht:
In the time frame of this thesis part of the obtained results have been published in the
following publications:

Jae-Hun Jeoung and Holger Dobbek (2007) Ni,Fe-containing Carbon Monoxide
Dehydrogenase, Handbook of Metalloproteins, A. Messerschmidt (ed.), John Wiley &
Sons, DOI: 10.1002/0470028637.met213, Article in Online.

Jae-Hun Jeoung and Holger Dobbek (2007) Carbon Dioxide Activation at the Ni,Fe-
Cluster of Anaerobic Carbon Monoxide Dehydrogenase, Science (318), 1461-1464.

Jae-Hun Jeoung, Diana A. Pippig, Berta M. Martins, Nadine Wagener and Holger
Dobbek (2007) HTHP: A Novel Class of Hexameric, Tyrosine-coordinated Heme
Proteins, Journal of Molecular Biology (368), 1122-1131.




















ACKNOWLEDGEMENTS
First, I would like to thank my supervisor, Prof. Dr. Holger
Dobbek, for giving me the opportunity to work with this fantastic
topic and for having confidence in me. I am grateful for his
enthusiastic and friendly supervision during the past four years. I
am greatly indebted to him for the work we have carried out and
Bioinorganic Chemistry
the successes we have achieved.

I hereby thank for Prof. Dr. Dr. h.c. Robert Huber, FRS, Dr. Sofia Macieira and Mr.
Snežan Marinkovic for giving me the opportunity to work with them and for sharing their
experiences at the beginning of my research. My time at the Max Planck Institute of
biochemistry (Structure Research, February – June 2004) has been a wonderful and
rewarding experience. I am forever indebted to these people for their contributions to
my scientific growth over the years.

I wish to thank Dr. Berta Martins for her constant encouragement, support and
guidance, not only for the scientific work but also for the beginning of my life in
Martinsried and Bayreuth. I am also grateful for her proofreading of my thesis.

I would also like to acknowledge the former Diploma students, Diana Pippig (worked
with HTHP), Till Giese and Marlene Grünerwald (worked with CooC proteins) and
Christian Gerhold (worked with HGO). I wish them great success in their Ph. D. work.

I greatly thank Roman Jacob for helping in measurement and interpretation of CD
spectra.

I thank PD. Dr. Vitali Svetlitchnyi for helpful advise during E. coli cultivation and activity
measurements of CODH, and for his kind donation of C. hydrogenoformans cells.

I wish to acknowledge the entire Lab members of the Bioinorganic Chemistry team
(formerly Protein Crystallography) for their resources and for providing such a
supportive and inspiring work environment.
To my parents in Korea, I would like to thank them for their patience, encouragements
and endless supports during my life.

Last but not least I want to acknowledge my family: my wife Hea-Suk, my son Hyun-
Bin and my daughter Ye-Rin for their support, patience and love.





















CONTENTS i
CONTENTS
ABBREVIATIONS 1
ZUSAMMENFASSUNG 3
SUMMARY 5
INTRODUCTION 6
1. CHEMISTRY OF CARBON MONOXIDE AND CARBON DIOXIDE 6
1.1. CARBON MONOXIDE (CO) 6
1.2. C DIOXIDE (CO ) 7 2
2. CARBON MONOXIDE DEHYDROGENASES (CODHS) 9
2.1. MO-,CU-CONTAINING CODH 10
2.2. NI-,FE-CONTAINING 14
3. NICKEL ENZYMES AND NICKEL PROCESSING SYSTEMS 20
3.1. HYDROGENASE SYSTEM 22
3.2. UREASE SYSTEM 24
3.3. CODH AND ACETYL-COA SYNTHASE SYSTEMS 26
4. STRUCTURAL COOC HOMOLOGUES IN THE SIMIBI CLASS 28
5. AIM OF STUDY 32
MATERIALS AND METHODS 33
1. CHEMICALS 33
2. ANAEROBIC WORK 33
3. BACTERIAL STRAINS AND CULTURE MEDIA 34
3.1. CARBOXYDOTHERMUS HYDROGENOFORMANS Z-2901 34
3.2. ESCHERICHIA COLI AND CULTURE MEDIA 34
4. MOLECULAR BIOLOGY 34
4.1. CLONING AND MUTAGENESIS OF COOC1 35
4.2. CLONING AND MUTAGENESIS OF COOSII 36
5. HETEROLOGOUS EXPRESSION OF PROTEIN 36
5.1. EXPRESSION OF COOC1 36
5.2. EXPRESSION OF REC-CODHII 37
6. PURIFICATION OF PROTEIN 38
6.1. PURIFICATION OF COOC1 38 CONTENTS ii
6.2. PURIFICATION OF REC-CODHII 39
7. MEASUREMENTS OF ENZYMATIC ACTIVITIES 40
7.1. NTPASE ACTIVITIES OF COOC1 40
7.2. CO-OXIDATION/CO -REDUCTION ACTIVITIES OF REC-CODHII 40 2
40 7.2.1. CO-OXIDATION ACTIVITY
41 7.2.2. CO -REDUCTION ACTIVITY 2
8. SPECTROSCOPIC ANALYSIS 41
8.1. UV-VISIBLE SPECTROSCOPY 41
42 8.1.1. COOC1 SPECTRA
42 8.1.2. REC-CODHII SPECTRA
8.2. CIRCULAR DICHROISM (CD) SPECTRA OF COOC1 42
42 8.2.1. FAR-UV CD SPECTRA
43 8.2.2. THERMALLY INDUCED UNFOLDING CD SPECTRA
9. SPECTROSCOPIC DETERMINATION OF NICKEL-BINDING TO COOC1 43
10. OLIGOMERIC STATES ANALYSIS OF COOC1 44
11. CRYSTALLOGRAPHIC METHODS 45
11.1. CRYSTALLIZATION OF COOC1 AND REC-CODHII 45
11.2. MANIPULATION OF CRYSTALS 46
11.3. DATA COLLECTION 47
11.4. STRUCTURE DETERMINATION 47
11.5. STRUCTURE REFINEMENT AND EVALUTION 47
12. MISCELLANEOUS METHODS 48
12.1. DETERMINATION OF PROTEIN CONCENTRATION 48
12.2. SDS-PAGE 48
12.3. COMPUTER SOFTWARES 48
RESULTS 49
1. CLONING 49
1.1. CLONING OF COOC1 49
1.2. CLONING OF COOSII 49
2. PROTEIN EXPRESSION AND PURIFICATION 50
2.1. EXPRESSION AND PURIFICATION OF COOC1 50
2.2. EXPRESSION AND PURIFICATION OF REC-CODHII 52
3. ATPASE AND GTPASE ACTIVITIES OF COOC1 55
4. SPECTROSCOPIC C HARACTERIZATION OF WILD-TYPE AND CLUSTER C-
MISSING REC-CODHII 57
5. SPECTROSCOPIC D ETERMINATION OF NI-BINDING AND NI-TITRATION OF
COOC1 58
CONTENTS iii
6. FAR-UV CD SPECTRA AND THERMAL UNFOLDING TRANSITIONS FOLLOWED
BY CD OF COOC1 61
7. OLIGOMERIC STATES OF COOC1 STUDIED BY GEL FILTRATION 62
8. CRYSTALLIZATION 64
8.1. CRYSTALLIZATION OF COOC1 64
8.2. CRYSTALLZATION OF REC-CODHII 65
9. STRUCTURES OF REC-CODHII 65
9.1. OVERALL STRUCTURE OF REC-CODHII 65
9.2. STRUCTURES OF THREE-REDOX STATES IN REVERSIBLE CO -REDUCTION 65 2
9.3. PROTON CHANNEL MUTANT H96D AND WATER NETWORK IN REC-CODHII 69
9.4. STRUCTURE OF CLUSTER C-MISSING REC-CODHII 71
10. STRUCTURES OF COOC1 72
10.1. OVERALL STRUCTURES 72
10.2. NUCLEOTIDE BINDING SITE 80
10.3. METAL BINDING SITE 84
DISCUSSION 87
1. CARBON DIOXIDE ACTIVATION AT THE NI,FE-CLUSTER OF ANAEROBIC CO
DEHYDROGENASE 87
2. SUBSTRATE AND PRODUCT T RANSFER P ATHWAYS IN CO
D 92
3. IMPLICATIONS OF CLUSTER C-MISSING CO DEHYDROGENASE 93
4. COOC1 IS AN ATPASE 94
5. COOC1 IS A NICKEL-BINDING PROTEIN 95
6. COOC1 UNDERGOES NI- AND NUCLEOTIDE-DEPENDENT DIMERIZATION 98
7. FUNCTIONAL MODEL FOR NI-PROCESSING OF COOC1 100
OUTLOOK 103
REFERENCES 104
APPENDIX 116
1. APPENDIX TABLES 116
2. APPENDIX FIGURES 122
CONTENTS iv
ERKLÄRUNG (DECLARATION) 132