Cryo-electron microscopy studies on ovine mitochondrial complex I

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Fiedorczuk, Karol 

The main objective of this work is to determine the atomic structure of mammalian respiratory complex I. Mitochondrial complex I (also known as NADH:ubiquinone oxidoreductase) is one of the central enzymes in the oxidative phosphorylation pathway. It couples electron transfer between NADH and ubiquinone to proton translocation across the inner mitochondrial membrane, contributing to cellular energy production. Complex I is the largest and most elaborate protein assembly of the respiratory chain with a total mass of 970 kilodaltons. It consists of 14 conserved ‘core subunits’ and 31 mitochondria-specific ‘supernumerary subunits’. Together they form a giant, Lshaped molecule, with one arm buried in the mitochondrial membrane and another protruding into the mitochondrial matrix. Here, a novel method for the purification of ovine (Ovis aries) complex I was developed and suitable conditions for cryo-EM imaging established, after extensive screening of detergents and additives. Cryo-EM images were acquired with the recently developed direct electron detector and processed using the latest software. This allowed the solution of the nearly complete atomic model of mitochondrial complex I at 3.9 Å resolution. The membrane part of the complex contains 78 transmembrane helices, mostly contributed by conserved antiporter-like subunits responsible for proton translocation. These helices are stabilized by tightly bound lipids (including cardiolipins). The hydrophilic arm harbours flavin mononucleotide and 8 iron–sulfur clusters involved in electron transfer. Supernumerary subunits build a scaffold around the conserved core, strongly stabilizing the complex. Additionally, subunits containing cofactors (NADPH, zinc ion and phosphopantetheine) may play a regulatory role. Two distinct conformations of the complex are observed, which may describe the active and deactive states or reflect conformations occurring during the catalytic cycle of the enzyme. Currently this is the most detailed model of this molecular machine, providing insight into the mechanism, assembly and dysfunction of mitochondrial complex I. It also allows molecular analysis of numerous disease-causing mutations, and so the structure may serve as a stepping-stone for future medical developments.

Sazanov, Leonid
mitochondria, complex I, respiration chan, Cryo-EM
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge