Biochemical and biophysical characterization of the human ethylmalonic encephalopathy non-heme sulfur (Fe)-dioxygenase ETHE1, and X-ray absorption spectroscopy applications and methods development [Elektronische Ressource] / presented by Marco Salomone-Stagni

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Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Science presented by Diploma-Biology (Laurea), Marco Salomone-Stagni Born in: Udine, Italy Oral-examination: 11.11.2010 Biochemical and Biophysical Characterization of the Human Ethylmalonic Encephalopathy non-Heme Sulfur [Fe]-Dioxygenase ETHE1, and X-ray Absorption Spectroscopy Applications and Methods Development Referees: Prof. Dr. Irmgard Sinning Dr. Andreas Ladurner To my parents and friends Preface This Thesis is based on the results presented in the following manuscripts: 1. Dominik Barthelme, Urte Scheele, Stephanie Dinkelaker, Adam Janoschka, Fraser MacMillan, Sonja-Verena Albers, Arnold J. M. Driessen, Marco S. Stagni, Eckhard Bill, Wolfram Meyer-Klaucke, Volker Schünemann and Robert Tampè Structural organization of essential iron-sulfur clusters in the evolutionarily highly conserved ATP-binding cassette protein ABCE1 Journal of Biological Chemistry, 2007 May 11; 282(19): 14598-607 2.

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Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Science













presented by

Diploma-Biology (Laurea), Marco Salomone-Stagni
Born in: Udine, Italy
Oral-examination: 11.11.2010



































Biochemical and Biophysical Characterization of the Human
Ethylmalonic Encephalopathy non-Heme Sulfur
[Fe]-Dioxygenase ETHE1, and X-ray Absorption Spectroscopy
Applications and Methods Development



















Referees: Prof. Dr. Irmgard Sinning
Dr. Andreas Ladurner




















































To my parents and friends







































Preface

This Thesis is based on the results presented in the following
manuscripts:

1. Dominik Barthelme, Urte Scheele, Stephanie Dinkelaker, Adam Janoschka,
Fraser MacMillan, Sonja-Verena Albers, Arnold J. M. Driessen, Marco S. Stagni,
Eckhard Bill, Wolfram Meyer-Klaucke, Volker Schünemann and Robert Tampè
Structural organization of essential iron-sulfur clusters in the evolutionarily
highly conserved ATP-binding cassette protein ABCE1
Journal of Biological Chemistry, 2007 May 11; 282(19): 14598-607

2. Seigo Shima, Oliver Pilak, Sonja Vogt, Marco S. Stagni, Wolfram Meyer-
Klaucke, Eberhard Warkentin, Rudolf K. Thauer, Ulrich Erlmer
The crystal structure of [Fe]-hydrogenase reveals the geometry of the active
site
Science, 2008 July 25; 321: 572-5

3. Takeschi Hiromoto, Kenichi Ataka, Oliver Pilak, Sonja Vogt, Marco S. Stagni,
Wolfram Meyer-Klaucke, Eberhard Warkentin, Rudolf K. Thauer, Seigo Shima,
Ulrich Ermler
The crystal structure of C176A mutated [Fe]-hydrogenase suggests an acyl-
iron ligation in the active site iron complex
Federation of European Biochemical Societies Letters, 2009 February 4; 583(3):
585-90

4. Stephan Binder, Marco Salomone-Stagni, Roxana Haase, Benjamin Schulz,
Andreas Eich, Gerald Henkel, Michael Rübhausen, Sonja Herres-Pawlis, and
Wolfram Meyer-Klaucke
I Characterization of the optically excited state of a bis (µ-oxo)-dicopper(III)
species mimicking the hemocyanin and tyrosinase active sites
Journal of Physics: Conference Series, 2009 November; 190; id: 012197

5. Marco Salomone-Stagni, Sonja Vogt, Rudolf K. Thauer, Seigo Shima, and
Wolfram Meyer-Klaucke
Extended X-ray absorption fine structure of the [Fe]-hydrogenase HMD
active site
Journal of Physics: Conference Series, 2009 November; 190; id: 012201

6. Marco Salomone-Stagni, Francesco Stellato, Matthew Whaley, Sonja Vogt,
Seigo Shima, Eckhard Bill, Thomas B. Rauchfuss, and Wolfram Meyer-Klaucke
The iron-site structure of [Fe]-hydrogenase and model systems: an X-ray
Absorption Near Edge Spectroscopy study
Dalton Transactions, 2010; 39: 3057-64. DOI: 10.1039/b922557a

7. Marco Salomone-Stagni, Alexey Kikhney, Eckhard Bill, and Wolfram Meyer-
Klaucke
Biochemical and biophysical characterization of the ethylmalonic
encephalopathy non-heme sulfur [Fe]-dioxygenase ETHE1 from Homo
sapiens
In preparation

Own contribution:
1. EXAFS and XANES analysis.
2. XAS measurements; EXAFS analysis.
3. XAS measurem
4. Pumped-XAS experimental set up, measurement and data evaluation;
contribution to the pilot resonance Raman spectroscopy data collection.
II

5. EXAFS analysis.
6. EXAFS analysis; XANES analysis.
7. Most of the work. I didn’t performed the Mössbauer data collection and analysis.
I contributed to the SAXS data analysis and evaluation.
III Table of contents
SUMMARY ...................................................................................................................... 1 
ZUSAMMENFASSUNG ................................................................................................. 2 
1 INTRODUCTION ........................................................................................................ 3 
1.1 Ethylmalonic encephalopathy protein 1 .............................................................................................. 3 
1.1.1 Homo sapiens ETHE1 ..................................................................................................................... 4 
1.1.2 HsETHE1 and hepatocellular carcinoma ......................................................................................... 5 
1.1.3 HsETHE1 and ethylmalonic encephalopathy .................................................................................. 5 
1.1.4 AtETHE1 structure ........................................................................................................................... 6 
1.1.5 HsETHE1 activity ............................................................................................................................ 9 
1.1.6 HsETHE1 metal binding site(s) ..................................................................................................... 10 
1.2 X-ray Absorption Spectroscopy (XAS) .............................................................................................. 12 
1.2.1 Mononuclear [Fe]-hydrogenases ................................................................................................... 16 
1.2.2 [Cu L (µ-O) ]I .............................................................................................................................. 18 2 2 2 2
1.2.3 The ATP binding cassette protein ABCE1 .................................................................................... 20 
1.3 Aim of the projects .............................................................................................................................. 21 
1.3.1 ETHE1 project ............................................................................................................................... 22 
1.3.2 XAS project ................................................................................................................................... 22 
2 MATERIALS AND METHODS ............................................................................... 23 
2.1 Cloning ................................................................................................................................................. 23 
2.1.1 Material for cloning ....................................................................................................................... 24 
2.1.1.1 Primers ................................................................................................................................... 24 
2.1.1.2 Kits used for cloning .............................................................................................................. 31 
2.1.1.3 Plasmid vectors ...................................................................................................................... 31 
2.1.1.4 Bacterial Strains ..................................................................................................................... 31 
2.1.1.5 Agarose Gel Electrophoresis (AGE) apparatus ...................................................................... 32 
2.1.1.6 Polymerase Chain Reaction (PCR) apparatus ........................................................................ 32 
2.1.2 Methods for cloning .................. 32 
2.1.2.1 Gene amplification ................................................................................................................. 32 
IV