The Mechanism of Proton Pumping by Respiratory Complex I

Saturday, February 11, 2017 various sub-complexes of the peripheral arm using recombinant subunits; and silenced the synthesis of certain subunits in vivo. Recently, an electron cryomicroscopy map of the enzyme was obtained. Putting together all the structural data available to date, we suggest a model of the complete topological disposition of the subunits that constitute the atypical, algal mitochondrial ATP synthase. The presence of such a stout peripheral stalk in the ATP synthase of chlorophycean alga, seems to have more than the necessary stiffness to counteract the rotation of the central stalk, but will probably make less twisting motions, and thus will store less transient elastic energy. 13-Subg The Mechanism of Proton Pumping by Respiratory Complex I Ulrich Brandt. Radboud Center for Mitochondrial Medicine, Nijmegen, Netherlands. Complex I (NADH:ubiquinone oxidoreductase) is the largest component of the respiratory chain. In mitochondria it contains one FMN and eight iron-sulfur clusters as prosthetic groups and is composed of some 40 subunits with a total mass of ~1,000 kDa. Human complex I deficiencies are the most frequent cause of inherited mitochondrial disorders and have been implicated in different neurodegenerative disorders and biological ageing. Complex I uses the energy released by the transfer of two electrons from NADH to ubiquinone to pump four protons across the bacterial plasma membrane or the inner mitochondrial membrane. The structures of complex I obtained by us and others from different species, now finally allows deep insight into the still obscure molecular mechanism of this process and its regulation by the so-called active/deactive transition. The ubiquinone chemistry taking place in the peripheral domain plays a pivotal role in this mechanism of energy conversion, by providing the energy that drives vectorial proton transport at the four putative pump sites in the membrane domain complex I. The two-state stabilization change mechanism of energy conversion by complex I is consistent with the functional and structural evidence now available. According to this comprehensive hypothetical model of energy conversion that will be presented in detail, the stepwise reduction and pronation of ubiquinone drives a concerted structural rearrangement, which exerts strokes passing ˚ to drive the proton pump into the membrane domain over a distance of ~200 A modules. The proposed mechanism is the first to inherently include thermodynamically feasible rationales for the reverse mode of the enzyme and its regulation by the active/deactive transition. Thereby, it has immediate implications for our understanding of different pathophysiological conditions involving mitochondrial function that will be discussed. 14-Subg Mitochondrial Metabolism Determines the Spatio-Temporal Organization of Single F1FO ATP Synthase in Live Human Cells Karin Busch. Institute for Molecular Cell Biology, Westfaelische Wilhelms University, Muenster, MUENSTER, Germany. Mitochondria are dynamic organelles on different levels. First, mitochondrial fusion and fission balance determines the morphology and dynamics of single organelles. Second, the internal structure can be re-organized following metabolic switches. Here, we investigate whether also on the protein level a dynamic adaption in terms of localization and dynamics occurs. By tracking and localization microscopy (TALM) we determine the dynamic organization of single F1FO ATP synthase in mitochondria under different conditions. It was shown before that mitochondria build stress induced hyperfused networks to escape mitophagy under starvation conditions. In parallel, b-oxidation of lipids in the matrix is enhanced. We asked, whether these changes are accompanied by a re-organization of mitochondrial substructures and whether and how this re-organization changes the spatio-temporal organization of proteins in the inner mitochondrial membrane. Indeed, we found that the spatio-temporal organization of mitochondrial F1FO ATP synthase was severely concerned. The mobility of F1FO ATP synthase was significantly enhanced and the localization more diffusive. We attribute this partially to ultrastructural changes since we found a strong reduction of the cristae membranes in starving cells. A switch towards higher OXPHOS rates by galactose supply had the same effect. Thus, the spatio-temporal organization of bioenergetics protein complexes on the molecular level is determined by metabolic conditions. 15-Subg Three-Dimensional Analysis of Human Mitochondrial Replicative Helicase Twinkle Maria Sola1, Pablo Ferna´ndez-Milla´n1, Melisa La´zaro2, Sirin Cansiz-Arda3, Joachim M. Gerhold3, Nina Rajala4, Claus A. Schmitz1, Cristina SilvaEspin˜a1, David Gil2, Pau Bernado´5, Mikel Valle2, Johannes N. Spelbrink3. 1 Structural Biology, Molecular Biology Institute of Barcelona (IBMB-CSIC), Barcelona, Spain, 2Structural Biology Unit, Centre for Cooperative Research

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in Biosciences (CICbioGUNE), Derio, Spain, 3Department of Pediatrics, Nijmegen Centre for Mitochondrial Disorders, Radboud University Medical Centre, Nijmegen, Netherlands, 4Mitochondrial DNA Maintenance Group, BioMediTech, University of Tampere, Tampere, Finland, 5Structural Biology, Centre de Biochimie Structurale, INSERM-U1054, CNRS UMR-5048, Universite´ de Montpellier I&II, Montpellier, France. The mitochondrial replication machinery includes essential proteins that are similar to T-odd bacteriophages, and thus its mechanism is different from the one in the nucleus. Involved proteins include the replicative helicase Twinkle, which unwinds the DNA at the replication fork. The threedimensional structure of Twinkle was solved by cryo-electron microscopy (cryo-EM) and small angle X-ray scattering (SAXS), which both showed the presence of heptamers and hexamers. Based on related helicase structures, we built an atomic homology model for the full-length protein, which included, for the first time for an SF4 helicase, an N-terminal zinc-binding domain (ZBD), an intermediate RNA polymerase domain (RPD), and a RecA-like hexamerization C-terminal domain (CTD). The cryo-EM structure of Twinkle reveals two hexameric rings, one comprising the ZBDs and RPDs and the other containing the CTDs. While the CTD ring shows strict six-fold symmetry, the ZBDs in the other ring show structural heterogeneity. We generated models in which the ZBD-RPD linker and the N-terminal segment of the RPD-CTD linker, as well as the N-terminal and the C-terminal tails, were defined as flexible regions with complete conformational freedom. From data measured in solution, we detected that both RPDs and ZBDs protrude considerably from the CTD hexa- and heptameric rings. The structural flexibility observed for the Twinkle N-terminal domains suggests a complex network of interactions consistent with functional requirements of helicase activity. 16-Subg Probing the Regulatory and Transport Mechanism of Mitochondrial Carriers with Thermostability Shift Assays Edmund Kunji. MRC Mitochondrial Biology Unit, Cambridge, United Kingdom. Mitochondrial carriers are membrane proteins that transport keto acids, amino acids, fatty acids, nucleotides, vitamins and inorganic ions across the inner membrane of mitochondria.1 Here we discuss the use of thermostability shift assays to probe the transport and regulatory mechanism of mitochondrial carriers. In these assays, protein unfolding is monitored with the fluorescent dye that emits a fluorescent signal upon reaction with cysteine residues.2 A controlled temperature ramp is used to denature the population of membrane proteins, exposing hidden cysteine residues to generate a melting curve.2,3 Here we show that they can be used to study the nature of the interactions of lipids, detergents, substrates and inhibitors with mitochondrial carriers and uncoupling proteins.3,4 In combination with mutagenesis they can also be used to show state-dependent amino acid interactions as part of a transport cycle5 and protein domain interactions as part of a regulatory mechanism. 1 Kunji ERS. Structural and Mechanistic Aspects of Mitochondrial Transport Proteins. In: Ferguson S, ed. Comprehensive Biophysics: Elsevier; 2012: 174-205. 2 Alexandrov AI, Mileni M, Chien EY, Hanson MA, Stevens RC. Microscale fluorescent thermal stability assay for membrane proteins. Structure 2008;16:351-9. 3 Crichton PG, Lee Y, Ruprecht JJ, et al. Trends in thermostability provide information on the nature of substrate, inhibitor, and lipid interactions with mitochondrial carriers. J Biol Chem 2015;290:8206-17. 4 Lee Y, Willers C, Kunji ER, Crichton PG. Uncoupling protein 1 binds one nucleotide per monomer and is stabilized by tightly bound cardiolipin. Proc Natl Acad Sci U S A 2015;112:6973-8. 5 King MS, Kerr M, Crichton PG, Springett R, Kunji ER. Formation of a cytoplasmic salt bridge network in the matrix state is a fundamental step in the transport mechanism of the mitochondrial ADP/ATP carrier. Biochim Biophys Acta 2016;1857:14-22. 17-Subg Pore Architecture and Ion Selectivity Filter of the Mitochondrial Calcium Uniporter James J. Chou. Dept Biochem/Molec Pharm, Harvard Med Sch, Boston, MA, USA. The calcium uniporter of mitochondria is a holocomplex consisting of the calcium-conducting channel, known as the MCU channel, and a score of regulatory proteins. Previous electrophysiology study found that a preeminent property of the uniporter is the high calcium selectivity and conductance and this has been shown to critically depend on the highly conserved amino acid