A Structural Perspective on Respiratory Complex I Leonid Sazanov Editor A Structural Perspective on Respiratory Complex I Structure and Function of NADH:ubiquinone oxidoreductase Editor Leonid Sazanov Medical Research Council Mitochondrial Biology Unit Wellcome Trust/MRC Building, Hills Road Cambridge CB2 0XY, UK ISBN 978-94-007-4137-9 ISBN 978-94-007-4138-6 (eBook) DOI 10.1007/978-94-007-4138-6 Springer Dordrecht Heidelberg New York London Library of Congress Control Number: 2012938257 © Springer Science+Business Media Dordrecht 2012 This work is subject to copyright. 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Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com) v Complex I (NADH:ubiquinone oxidoreductase) is the fi rst enzyme of the respira- tory chain in mitochondria and bacteria. It is one of the largest and most elaborate membrane protein assemblies known. It plays a central role in cellular energy pro- duction, providing about 40% of the proton fl ux required for ATP synthesis. Complex I dysfunction has been implicated in many human neurodegenerative diseases and mutations in its subunits are the most common human genetic disorders known. Complex I is also a major source of reactive oxygen species in mitochondria, which may lead to Parkinson’s disease and could be involved in aging. The enzyme transfers two electrons from NADH to quinone, coupling this process to the translocation of four protons across the membrane out of the mitochondrial matrix, by a mechanism as yet not fully established. Mitochondrial complex I consists of 45 different subunits, whilst the prokaryotic enzyme is simpler, consisting of 14 “core” subunits with a total mass of about 550 kDa. The mitochondrial and bacterial enzymes contain equivalent redox components ( fl avin and 8–9 Fe-S clusters) and have a similar, rather unusual, L-shaped structure. The hydrophobic arm is embedded in the mem- brane and the hydrophilic peripheral arm protrudes into the mitochondrial matrix or the bacterial cytoplasm. The “core” subunits exhibit a high degree of sequence conservation, which suggests that the complex I mechanism is likely to be the same throughout all species. Hence, the bacterial enzyme is used as a ‘minimal’ model of human complex I in order to understand its structure and mechanism. Recent years have been marked by spectacular progress in the structural characterization of complex I, which now fi nally allows us to begin to understand the mechanics of this large molecular machine, making this book very timely. Until about 5–6 years ago structural information on complex I was absent, and so understanding of it was very limited, especially compared to other enzymes of the respiratory chain. Complex I used to be known as a notorious “monster” enzyme, the “black box” of bioenergetics. In 40 or so years since its discovery it was esta- blished that complex I most likely pumps four protons per two electrons transferred from NADH to quinone. Electron transfer was known to occur via fl avin mononu- cleotide (FMN) and series of at least 6 iron-sulfur (Fe-S) clusters, which were detected by electron paramagnetic resonance (EPR). Not all the clusters were Foreword vi Foreword observed experimentally, since the presence of 8–9 Fe-S clusters was predicted on the basis of sequence analysis. The sequence of events during electron transfer was unknown and the mechanism of proton translocation was even more enigmatic. Two possible mechanisms of coupling between electron transfer and proton translocation have been vigorously discussed: direct (redox-driven, akin to the Q-cycle) and indirect (conformation-driven). However, in the absence of structural information, they were mostly speculative. All started to change in 2005–2006, when we solved the fi rst crystal structure of the hydrophilic domain of complex I, using the enzyme from Thermus thermophilus. It established the electron transfer pathway from NADH, through fl avin mononu- cleotide (FMN) and seven conserved Fe-S clusters, to the quinone-binding site at the interface with the membrane domain. In 2010–2011 we have solved the struc- ture of the membrane domain of E. coli complex I and determined the architecture of the entire T. thermophilus enzyme at lower resolution. Thus, the atomic structure of only one “core” subunit, Nqo8/NuoH ( Thermus/E. coli nomenclature), found at the interface of the two main domains, remains unknown. Additionally, low-resolution X-ray analysis of the mitochondrial enzyme from Yarrowia lypolityca was published in 2010, indicating a similar arrangement of the “core” subunits, surrounded by many supernumerary subunits. The membrane-spanning part of the enzyme lacks covalently bound prosthetic groups, but our structures show how proton translocation through the three largest hydrophobic subunits of complex I, homologous to each other and to the antiporter family, may be driven by a long a -helix, akin to the cou- pling rod in a steam engine. This and other features of the structure strongly suggest that electron transfer in the peripheral arm is coupled to proton translocation in the membrane arm purely by long-range conformational changes. Mutations causing human diseases are found near key residues involved in proton transfer, explaining their effects on activity. Not all the details of the mechanism are clear yet, but we are now operating on a completely different level of knowledge than just a few years ago. This led to the idea of summarizing in book form current knowledge of complex I, taking into account structural information. No books on complex I have been published previ- ously, and the last special issue of a journal devoted to complex I was published in 2001, when it was still known as a “black box”. Therefore, it is hoped that this book will provide the reader with a timely and comprehensive review of current state- of-the-art research on complex I. In Chap. 1 , current knowledge of the structure of complex I is reviewed, starting from the peripheral domain, followed by a detailed description of the new structure of the membrane domain, and ending with implications for the mechanism. In Chap. 2 , the binding of substrates, the role of individual Fe-S clusters (in particular those away from the main pathway) and the mechanism of proton translocation are discussed on the basis of data from site-directed mutagenesis, EPR and FTIR spectroscopy, as well as other studies. In Chap. 3 , current knowledge of the characteristics and roles of each Fe-S cluster in complex I is overviewed. Chapter 4 provides a review of many speci fi c inhibitors of complex I, the use of which has been very informative in characterisation of the quinone-binding site and the terminal electron transfer step. vii Foreword In Chap. 5 , some of the earliest studies on complex I, in particular EPR spectroscopy leading to the fi rst identi fi cation of Fe-S clusters, are summarised. Complex I has an intricate evolutionary history, originating from the uni fi cation of hydrogenase and transporter modules. In Chap. 6 , the evolutionary relationship with [Ni-Fe]-hydrogenases is analysed and mechanistic implications are derived from comparisons of known crystal structures. In Chap. 7 , the emphasis is on the relationship with the Mrp antiporter family and it is proposed that antiporter-like subunits in modern complex I may have different functions. Mutations in complex I subunits, both mitochondrially- and nuclear-encoded, lead to a range of human diseases. Many of these mutations have been reproduced in bacterial systems for mechanistic studies. Chapter 8 provides a review of site- directed mutagenesis studies that helped in identifying residues essential for structural integrity, cofactor ligation, substrate binding, electron transfer and proton translocation. In Chap. 9 , a comprehensive overview of the cellular consequences of pathological mtDNA-encoded mutations in complex I subunits is provided. Mitochondrial complex I contains, in addition to the “core” subunits, up to 31 “supernumerary” subunits, with poorly understood roles. Chapter 10 describes an intricate process of assembly of the complex in several stages, involving distinct func- tionally and evolutionarily conserved modules, and requiring a number of chaperones. In Chap. 11 , the similarities and peculiarities of the subunit composition of mitochon- drial complex I in plants and the complex I analogue in chloroplasts are described. In the respiratory chain of mitochondria complex I appears not to exist on its own, but as part of even larger assemblies, or “supercomplexes”. These involve complexes I, III and IV, as described in Chap. 11 , and may promote substrate channelling. Thus, combined, the chapters cover a wide range of topics which should provide the reader with an up-to-date review of research on complex I in these exiting times, when the molecular basis for its mechanism is fi nally starting to become clear. Leonid Sazanov Medical Research Council Mitochondrial Biology Unit Wellcome Trust/MRC Building, Hills Road Cambridge, UK ix Part I Structure and Mechanism of Complex I 1 Structure of Complex I 3 Rouslan G. Efremov and Leonid Sazanov 2 On the Mechanism of the Respiratory Complex I 23 Thorsten Friedrich, Petra Hellwig, and Oliver Einsle 3 Iron–Sulfur Clusters in Complex I 61 Eiko Nakamaru-Ogiso 4 Current Topics of the Inhibitors of Mitochondrial Complex I 81 Hideto Miyoshi 5 My Fifty Years Association with Complex I Study 99 Tomoko Ohnishi Part II Evolution of Complex I 6 The Evolutionary Relationship Between Complex I and [NiFe]-Hydrogenase 109 Anne Volbeda and Juan C. Fontecilla-Camps 7 Recruitment of the Antiporter Module – A Key Event in Complex I Evolution 123 Vamsi Krishna Moparthi and Cecilia Hägerhäll Part III Mutations in Complex I Subunits and Medical Implications 8 Characterization of Bacterial Complex I (NDH-1) by a Genetic Engineering Approach 147 Takao Yagi, Jesus Torres-Bacete, Prem Kumar Sinha, Norma Castro-Guerrero, and Akemi Matsuno-Yagi Contents x Contents 9 Cellular Consequences of mtDNA-Encoded Mutations in NADH:Ubiquinone Oxidoreductase 171 Mina Pellegrini, Jan A.M. Smeitink, Peter H.G.M. Willems, and Werner J.H. Koopman Part IV Subunit Composition and Assembly of Mitochondrial Complex I 10 The Assembly of Human Complex I 193 Jessica Nouws, Maria Antonietta Calvaruso, and Leo Nijtmans 11 Complexes I in the Green Lineage 219 Claire Remacle, Patrice Hamel, Véronique Larosa, Nitya Subrahmanian, and Pierre Cardol Part V Supercomplexes in Mitochondria 12 Supramolecular Organization of the Respiratory Chain 247 Janet Vonck A Structural Perspective on Complex I 279 Index 281 [...]... likely indirectly driven by the (ubi)quinone redox chemistry Surprisingly, one of the ATS contains a long horizontal helix aligning the membrane arm It was suggested that this amphipatic helix acts as a ‘piston’ transmitting the energy released by the redox reaction to the ATS (Efremov et al 2010; Ohnishi 2010) Bacteria contain a structurally minimal form of an energy-converting NADH:ubiquinone oxidoreductase... 2003) and possibly aging (Balaban et al 2005) Mutations in nucleus and mitochondria encoded subunits have been associated with several neurodegenerative diseases (Sazanov 2007; Schapira 1998) Complex I has an intricate evolutionary history, representing a chimera of hydrogenases and cation-proton antiporters (reviewed in (Friedrich 2001; Moparthi and Hagerhall 2011)) The complex is present in many bacteria... fast-relaxing semiquinone (QNf), sensitive to the membrane potential and interacting with cluster N2, and slow-relaxing semiquinone (QNs), insensitive to trans-membrane potential and not interacting with N2 (Ohnishi 1998; Ohnishi et al 2010b) Additionally, mutations of GluTM5 in NuoN do not affect activity as drastically as those in NuoL and M (Amarneh and Vik 2003) and, as noted above (Mathiesen and... translocation of four protons across the inner mitochondrial membrane (in eukaryotes) or cytoplasmic membrane (in bacteria), with a maximum rate of about 200 cycles per second (Walker 1992; Yagi and Matsuno-Yagi 2003; Sazanov 2007; Brandt 2006) It is also considered as the main source of reactive oxygen species (ROS) in mitochondria, which can damage mtDNA and cause Parkinson’s disease (Dawson and Dawson... Coupling and Proton-Pumping Mechanisms Advances in resolving high-resolution structure allow us now to comprehend many controversial aspects of the mechanism of complex I, although raising simultaneously new questions In combination, all the structural features indicate unambiguously that complex I operates purely by a conformation-driven mechanism Based on the available structural data the following... and are parts of putative proton translocation channels Only MHis241 and NHis224 (structurally and sequentially conserved) are located on TM7 pointing outside the subunit However, they interact directly with helix HL, which is likely the primary reason for their conservation Importantly, inhibition or lack of activation with decylubiquinone were observed for mutations of other surface residues interacting... mechanism Electron microscopic reconstructions of the enzyme structure in negative stain and in vitreous ice established its overall L-shaped appearance in all organisms studied (Clason et al 2010), with a peripheral arm protruding into the bacterial cytoplasm/ mitochondrial matrix and a membrane embedded arm The mass of the enzyme is approximately equally distributed between peripheral and membrane arms,... and mechanism of a Na+-independent amino acid transporter Science 325:1010–1014 Shinzawa-Itoh K, Seiyama J, Terada H, Nakatsubo R, Naoki K, Nakashima Y, Yoshikawa S (2010) Bovine heart NADH-ubiquinone oxidoreductase contains one molecule of ubiquinone with ten isoprene units as one of the cofactors Biochemistry 49:487–492 Sled VD, Rudnitzky NI, Hatefi Y, Ohnishi T (1994) Thermodynamic analysis of flavin... discussed as potential quinone binding sites M (Fisher and Rich 2000; Amarneh and Vik 2003; Nakamaru-Ogiso et al 2010b) 14 R.G Efremov and L Sazanov Third, Amarneh and Vik (2003) observed inhibition of NADH oxidase activity by decylubiquinone in several mutants, including NHis224 The structure shows that the majority of the above mentioned histidines are, in fact, buried deep inside the protein and... membrane and the position of the (ubi) quinone binding site(s) remain under debate, it became clear that most redox reactions do not contribute to proton translocation It is now evident that only the (ubi) quinone chemistry, possibly assisted by the redox reaction of N2 drives proton translocation by both a direct and an indirect mechanism The membrane arm is composed of seven ‘minimal’ subunits Among . Hägerhäll Part III Mutations in Complex I Subunits and Medical Implications 8 Characterization of Bacterial Complex I (NDH-1) by a Genetic Engineering Approach 147 Takao Yagi, Jesus Torres-Bacete,. mitochondria, which can damage mtDNA and cause Parkinson’s disease (Dawson and Dawson 2003 ) and possibly aging (Balaban et al . 2005 ) . Mutations in nucleus and mitochon- dria encoded subunits have. A Structural Perspective on Respiratory Complex I Leonid Sazanov Editor A Structural Perspective on Respiratory Complex I Structure and Function of NADH:ubiquinone oxidoreductase Editor Leonid