The ubiquinone and inhibitor binding pocket of complex I from Yarrowia lipolytica [Elektronische Ressource] : a structure-based mutagenesis study / von Maja Aleksandra Tocilescu

The Ubiquinone and Inhibitor Binding Pocket
of Complex I from Yarrowia lipolytica: Thesis
A Structure-based Mutagenesis Study





by
Maja Aleksandra Tocilescu
Molecular Bioenergetics Group
Goethe-University Medical School
Frankfurt am Main


















Cover picture was adapted from (Zickermann et al., 2009).
For details see Summary Figure 5.1.








The Ubiquinone and Inhibitor Binding Pocket
of Complex I from Yarrowia lipolytica:
A Structure-based Mutagenesis Study


Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften


vorgelegt beim Fachbereich Biowissenschaften
der Johann Wolfgang Goethe - Universität
in Frankfurt am Main


von
Maja Aleksandra Tocilescu
aus Belgard, Polen


Frankfurt 2009
(D 30)










vom Fachbereich Biowissenschaften der Johann Wolfgang Goethe - Universität als
Dissertation angenommen.


Dekan : Prof. Dr. Volker Müller


Gutachter : Prof. Dr. Volker Müller

Prof. Dr. Ulrich Brandt


Datum der Disputation : 8. Mai 2009





Die vorliegende Arbeit wurde im Labor von Prof. Dr. Ulrich Brandt, Arbeitsgruppe
Molekulare Bioenergetik am Zentrum der Biologischen Chemie des Fachbereichs
Medizin der Johann Wolfgang Goethe-Universität, Frankfurt am Main im Zeitraum
vom August 2005 bis Februar 2009 angefertigt.

















Index of Contents I

Index of Contents
1  Introduction ..................................................................................... 1 
1.1  The mitochondrial respiratory chain .............................................................. 1 
1.2  Complex I .......................................................................................................... 3 
1.2.1  Subunit composition ...................................................................................................... 3 
1.2.2  Structural data ................................................................................................................ 4 
1.2.3  The ubiquinone binding site .......................................................................................... 6 
1.2.4  Complex I inhibitors 7 
1.2.5  EPR spectroscopy of iron-sulfur clusters....................................................................... 9 
1.2.6  Iron-sulfur cluster N1a ................................................................................................. 13 
1.2.7  The mechanism of energy conversion ......................................................................... 13 
1.3  Yarrowia lipolytica as a model organism ....................................................... 14 
1.4  Aim of this study ............................................................................................. 17 
1.4.1  Exploring the quinone and inhibitor binding pocket ................................................... 17 
1.4.2  Characterization of the HRGXE-motif ........................................................................ 18 
1.4.3  Shifting of the midpoint potential of iron-sulfur cluster N1a ...................................... 19 
1.4.4  Developing a new in vivo screen for complex I deficiency ......................................... 19 
2 Materials and Methods ................................................................. 21
2.1 Materials .......................................................................................................... 21
2.1.1  Chemicals .................................................................................................................... 21 
2.1.2  Inhibitors ...................................................................................................................... 22 
2.1.3  Enzy m es ....................................................................................................................... 23 
2.1.4  Plasmids ....................................................................................................................... 23 
2.1.5  Strains .......................................................................................................................... 24 
2.1.6  Instruments .................................................................................................................. 25 
2.1.7  Software ....................................................................................................................... 27 
2.2  Methods ........................................................................................................... 28 
2.2.1  Methods of Microbiology ............................................................................................ 28 
2.2.2 Molecular Biology .................................................................................... 30 
2.2.3  Methods of Protein Chemistry ..................................................................................... 33 
2.2.4  Methods of Bioinformatics .......................................................................................... 38 II Index of Contents
3  Results ............................................................................................ 41 
3.1  Mutagenesis of the proposed ubiquinone and inhibitor binding cavity ..... 41 
3.1.1  Assembly of mutant complex I .................................................................................... 41 
3.1.2  Activity of mutant complex I ....................................................................................... 49 
3.1.3  Apparent K for DBQ .................................................................................................. 52 m
3.1.4  Complex I activity with Q and Q as electron acceptors ............................................ 52 1 9
3.1.5  I values for DQA, rotenone and C E ....................................................................... 55 50 12 8
3.1.6  EIPA ............................................................................................................................. 61 
3.2  In vivo screen for complex I deficiency ......................................................... 66 
3.2.1  The principle ................................................................................................................ 66 
3.2.2  Development of a plate assay for complex I activity ................................................... 67 
3.2.3  Screening of complex I mutants................................................................................... 69 
3.3  Tyrosine 144 ..................................................................................................... 71 
3.3.1  Assembly and activity of tyrosine 144 mutants ........................................................... 72 
3.3.2  Iron-sulfur cluster N2 in tyrosine 144 mutants ............................................................ 73 
3.3.3  Complex I activity at higher DBQ concentrations in mutant Y144F ........................... 78 
3.3.4  pH dependence of residual activity of mutant Y144F ................................................. 79 
3.3.5  Reactivation of mutant Y144F ..................................................................................... 81 
3.3.6  dNADH:Q oxidoreductase activity in tyrosine 144 mutants ...................................... 85 1
3.3.7 Q oxidoreductase activity of mutants Y144F and Y144W .......................... 87 2
3.3.8  Inhibitor resistance of the tyrosine 144 mutants .......................................................... 88 
3.3.9  Proton pumping of complex I in mutants Y144F and Y144W .................................... 89 
3.4  Mutagenesis study of the HRGXE-motif ...................................................... 92 
3.4.1  The HRGXE-motif in different organisms .................................................................. 92 
3.4.2  Location of the HRGXE-motif .................................................................................... 93 
3.4.3  Complex I assembly and activity of the HRGXE-motif mutants ................................ 94 
3.4.4  EPR spectra from mutants of the HRGXE-motif ......................................................... 95 
3.4.5  Apparent K and I values for mutants of the HRGXE-motif .................................... 97 m 50
3.5  Mutagenesis of the 24-kDa subunit close to iron-sulfur cluster N1a .......... 97 
3.5.1  Selection of targeted residues ................