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Complex I)’ Is the Result of My Own Work and Includes Nothing Which Is the Outcome of Work Done in Collaboration Except Where Specifically Indicated in the Text

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Complex I)’ Is the Result of My Own Work and Includes Nothing Which Is the Outcome of Work Done in Collaboration Except Where Specifically Indicated in the Text Declaration This thesis, entitled ‘structural and functional studies of mitochondrial NADH:ubiquinone oxidoreductase (complex I)’ is the result of my own work and includes nothing which is the outcome of work done in collaboration except where specifically indicated in the text. This work was carried out between October 2006 and July 2010 at the MRC Mitochondrial Biology Unit under the supervision of Dr. Judy Hirst. Information derived from other sources has been referenced accordingly. Martin S. King August, 2010 i Paul William Brady 16 th February 1984 - 18 th January 2008 Life ain’t always what it seems to be, Words can’t express what you mean to me, Even though you’re gone, we’re still a team, Through your family, I’ll fulfil your dreams, In the future, can’t wait to see, If you open up the gates for me, Reminisce some time, the night they took my friend, Try to black it out, but it plays again, When it’s real, feelings hard to conceal, Can’t imagine all the pain I feel, Give anything to hear half your breath, I know you’re still living your life, after death. “I’ll Be Missing You” Puff Daddy and Faith Evans ii Acknowledgements First and foremost, I must thank Dr. Judy Hirst for putting up with me for four years; her enthusiasm and patience never faltered, even when I was banging my head against a brick wall. Dr. Hirst’s endless knowledge of all things complex I proved invaluable, without which, I would have ended up very lost. Thanks must also go to other members of the Hirst lab, particularly those who have proofread this thesis. A special thanks to Drs. Steve Sherwood and Hannah Bridges, whose fresh ideas and solutions to my mundane problems kept me sane. I also wish to thank other members of the Mitochondrial Biology Unit for their advice, assistance and crystal- shooting during my PhD, especially Martin Montgomery and Mike Runswick, and Drs. John Berrisford, Rouslan Efremov, Edmund Kunji and Jonathan Ruprecht. Our collaborators also deserve my thanks, especially Maxie Roessler at Oxford. Thanks also to my numerous housemates over the course of the past four years: Lizzie Fellowes-Freeman, Heeran Buhecha, Heather Burgess, Helen Craig, David Wyatt and Hayley King have all put up with my misanthropic view of human nature, my warped sense of humour, and my awful guitar ‘playing’. I must also show my appreciation for the MRC, who funded my research, as well as the fellows and staff of St. Catharine’s College for their ‘free’ food over the course of my PhD. In addition, I must thank Arsenal Football Club who, without fail, has proved to be a constant source of heartache and frustration, and Chandler Bing and Gregory House, M.D., my role models, my heroes. Finally, I cannot put into words how much I am indebted to my family. Quite simply, I would not be where I am today if it were not for the love and support of my Mum, Patricia, my Dad, Stephen (yes, that’s Stephen King…) and my sister, Hayley. - This thesis is dedicated to my family, and to the memory of Paul Brady - iii Abstract NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme in the mitochondrial electron transfer chain. It catalyses the oxidation of NADH and the reduction of ubiquinone, coupled to the translocation of protons across the mitochondrial inner membrane, maintaining the proton motive force used for ATP synthesis. Complex I is the least understood of the respiratory enzymes; although the mechanisms of NADH oxidation and intramolecular electron transfer are gradually becoming appreciated, the mechanisms of quinone binding and reduction and proton translocation remain unknown. Complex I dysfunction has been implicated in a wide range of pathologies including mitochondrial diseases such as Leigh’s disease, as well as neurodegenerative diseases such as Alzheimer’s and Parkinson’s. The work described in the first part of this thesis is aimed at elucidating the structure of either a subcomplex of mitochondrial complex I, or of the intact enzyme itself. A comprehensive investigation revealed that hydrophilic subcomplexes of complex I from bovine heart mitochondria are not suitable for use as models of the intact enzyme. Attempts to prepare intact complex I of sufficient quality for structural work were successful; however, results from a large set of crystallization trials were disappointing. The second part of this thesis describes three studies of the function and mechanism of complex I from bovine heart mitochondria. First, the flavin mononucleotide, the site of NADH oxidation, was identified as the site of the ‘inhibitor-insensitive’ NADH:ubiquinone oxidoreduction reaction. The formation of semiquinones initiates redox cycling reactions with oxygen, producing vast amounts of reactive oxygen species; further studies revealed that other oxidants, such as paraquat, also react at the flavin site and initiate redox cycling reactions. Second, kinetic studies showed that the reaction between NADH and positively charged oxidants such as HAR (hexaammineruthenium (III)) proceeds by an unusual ternary reaction mechanism at the flavin site of complex I. Finally, double electron-electron resonance spectroscopy was used to show unambiguously that iron sulphur cluster 4Fe[TY]1 gives rise to electron paramagnetic resonance signal N4; the data provide an alternating potential energy profile for electron transfer along the cluster chain between the flavin and the quinone-binding site. iv Abbreviations 5C Cymal-5 6C Cymal-6 7C Cymal-7 8G/OG n-octyl-β-D-glucopyranoside 10M/DM n-decyl-β-D-maltopyranoside 11M/UDM n-undecyl-β-D-maltopyranoside 12M/DDM n-dodecyl-β-D-maltopyranoside APAD + 3-acetylpyridine adenine dinucleotide ADP Adenosine diphosphate ADP-ribose Adenosine 5 ′-diphosphoribose AR Amplex red Aso Asolectin ATP Adenosine triphosphate β Bohr magneton: 9.274 x 10 -24 J T -1 B. taurus Bos taurus BCA Bicinchoninic acid BSA Bovine serum albumin Cardio Cardiolipin CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate Complex I NADH:ubiquinone oxidoreductase Complex II Succinate:ubiquinone oxidoreductase Complex III Ubiquinone:cytochrome c oxidase Complex IV Cytochrome c oxidase CMC Critical micelle concentration CMT Critical micelle temperature Cyt c Partially acetylated cytochrome c DEER Double electron electron resonance ∆p Proton motive force ∆pH pH on the positive side (the inter membrane space) minus the pH on the negative side (the mitochondrial matrix) ∆ψ Membrane potential DOPC Dioleolylphosphatidylcholine v DPI Diphenyleneiodonium DQ Decylubiquinone DTAB Dodecyl-trimethylammonium bromide DTT Dithiothreitol ε Molar extinction coefficient E. coli Escherichia coli EDTA Ethylenediaminetetraacetic acid EPR Electron paramagnetic resonance ETF Electron-transferring flavoprotein ETF:Q Electron-transferring flavoprotein:ubiquinone oxidoreductase F Faraday constant: 9.65 x 10 -2 kJ mol -1 mV -1 FAD Flavin adenine dinucleotide FeCN Ferricyanide/potassium hexacyanoferrate (III) FMN Flavin mononucleotide FDRA Friedreich’s Ataxia G Gauss GPX Glutathione peroxidase GR Glutathione reductase GSH/GSSG Reduced/oxidised glutathione h Planck’s constant: 6.626 x 10 -34 J s H Magnetic field HAR Hexaammineruthenium (III) chloride HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HRP Horse radish peroxidase HPLC High performance liquid chromatography Iα Subcomplex I α Iβ Subcomplex I β Iγ Subcomplex I γ Iλ Subcomplex I λ IλDTAB Subcomplex I λ without subunit B16.6 (after treatment with DTAB) IMS Inter-membrane space LDAO Lauryldimethylamine-oxide LSQE Least-squares error MBU Mitochondrial Biology Unit vi MGDG Monogalactosyldiacyl glycerol MES 2-(N-morpholino)ethanesulfonic acid MOPS 3-(N-morpholino)propanesulfonic acid MPDB Membrane protein data bank (www.mpdb.tcd.ie/) MRC Medical Research Council mtDNA Mitochondrial DNA MWCO Molecular weight cut-off N. crassa Neurospora crassa NAD + Nicotinamide adenine dinucleotide NAD(P) + Nicotinamide adenine dinucleotide phosphate NADH Reduced nicotinamide adenine dinucleotide NAD(P)H Reduced nicotinamide adenine dinucleotide phosphate O. aries Ovis aries P. denitrificans Paracoccus denitrificans P. pastoris Pichia pastoris PAGE Polyacrylamide gel electrophoresis PC Phosphatidylcholine PDB Protein data bank (www.pdb.org) PE Phosphatidylethanolamine PEG x Polyethylene glycol, average molecular weight of x Pxr Peroxiredoxin Pier Piericidin A PMSF Phenylmethylsulphonyl fluoride PPM Parts per million Q0 Coenzyme Q 0 / ubiquinone-0 Q1 Coenzyme Q 1 / ubiquinone-1 Q2 Coenzyme Q 2 / ubiquinone-2 Q10 Coenzyme Q 10 / ubiquinone-10 R. capsulatus Rhodobacter capsulatus R Gas constant: 8.314 J K -1 mol -1 Rot Rotenone ROS Reactive oxygen species S. cerevisiae Saccharomyces cerevisiae S. scrofa Sus scrofa vii SDS Sodium dodecyl sulphate SMP Sub-mitochondrial particle SOD Superoxide dismutase T Temperature T. thermophilus Thermus thermophilus TCEP Tris (2-carboxyethyl) phosphine TEMED N, N, N', N' - tetramethylethylene diamine Tris Tris (hydroxymethyl) aminomethane UV Ultraviolet v Microwave frequency QH 2 Ubiquinol v/v Volume by volume w/v Weight by volume w/w Weight by weight Y. lipolytica Yarrowia lipolytica viii Contents Title Page.................................................................................................................. Declaration ............................................................................................................... i In memoriam............................................................................................................ ii Acknowledgements
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