Browsing by Subject "MEMBRANE DOMAIN"

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  • Yoga, Etienne Galemou; Haapanen, Outi; Wittig, Ilka; Siegmund, Karin; Sharma, Vivek; Zickermann, Volker (2019)
    Respiratory complex I catalyses the reduction of ubiquinone (Q) from NADH coupled to proton pumping across the inner membrane of mitochondria. The electrical charging of the inner mitochondrial membrane drives the synthesis of ATP, which is used to power biochemical reactions of the cell. The recent surge in structural data on complex I from bacteria and mitochondria have contributed to significant understanding of its molecular architecture. However, despite these accomplishments, the role of various subdomains in redox-coupled proton pumping remains entirely unclear. In this work, we have mutated conserved residues in the loop of the PSST subunit that faces the similar to 30 angstrom long unique Q-binding tunnel of respiratory complex I. The data show a drastic decrease in Q reductase activity upon mutating several residues despite full assembly of the complex. In-silico modeling and multiple microsecond long molecular dynamics simulations of wild-type and enzyme variants with exchanges of conserved arginine residues revealed remarkable ejection of the bound Q from the site near terminal electron donor N2. Based on experiments and long-time scale molecular simulations, we identify microscopic elements that dynamically control the diffusion of Q and are central to redox-coupled proton pumping in respiratory complex I.
  • Djurabekova, Amina; Haapanen, Outi; Sharma, Vivek (2020)
    In the aerobic respiratory chains of many organisms, complex I functions as the first electron input. By reducing ubiquinone (Q) to ubiquinol, it catalyzes the translocation of protons across the membrane as far as ~200 Å from the site of redox reactions. Despite significant amount of structural and biochemical data, the details of redox coupled proton pumping in complex I are poorly understood. In particular, the proton transfer pathways are extremely difficult to characterize with the current structural and biochemical techniques. Here, we applied multiscale computational approaches to identify the proton transfer paths in the terminal antiporter-like subunit of complex I. Data from combined classical and quantum chemical simulations reveal for the first time structural elements that are exclusive to the subunit, and enables the enzyme to achieve coupling between the spatially separated Q redox reactions and proton pumping. By studying long time scale protonation and hydration dependent conformational dynamics of key amino acid residues, we provide novel insights into the proton pumping mechanism of complex I.
  • Warnau, Judith; Sharma, Vivek; Gamiz-Hernandez, Ana P.; Di Luca, Andrea; Haapanen, Outi; Vattulainen, Ilpo; Wikström, Mårten; Hummer, Gerhard; Kaila, Ville R. I. (2018)
    Complex I couples the free energy released from quinone (Q) reduction to pump protons across the biological membrane in the respiratory chains of mitochondria and many bacteria. The Q reduction site is separated by a large distance from the proton-pumping membrane domain. To address the molecular mechanism of this long-range proton-electron coupling, we perform here full atomistic molecular dynamics simulations, free energy calculations, and continuum electrostatics calculations on complex I from Thermus thermophilus. We show that the dynamics of Q is redox-state-dependent, and that quinol, QH(2), moves out of its reduction site and into a site in the Q tunnel that is occupied by a Q analog in a crystal structure of Yarrowia lipolytica. We also identify a second Q-binding site near the opening of the Q tunnel in the membrane domain, where the Q headgroup forms strong interactions with a cluster of aromatic and charged residues, while the Q tail resides in the lipid membrane. We estimate the effective diffusion coefficient of Q in the tunnel, and in turn the characteristic time for Q to reach the active site and for QH2 to escape to the membrane. Our simulations show that Q moves along the Q tunnel in a redox-state-dependent manner, with distinct binding sites formed by conserved residue clusters. The motion of Q to these binding sites is proposed to be coupled to the proton-pumping machinery in complex I.
  • Haapanen, Outi; Sharma, Vivek (2017)
    Membrane bound respiratory complex I is the key enzyme in the respiratory chains of bacteria and mitochondria, and couples the reduction of quinone to the pumping of protons across the membrane. Recently solved crystal or electron microscopy structures of bacterial and mitochondrial complexes have provided significant insights into the electron and proton transfer pathways. However, due to large spatial separation between the electron and proton transfer routes, the molecular mechanism of coupling remains unclear. Here, based on atomistic molecular dynamics simulations performed on the entire structure of complex I from Thermus thermophilus, we studied the hydration of the quinone-binding site and the membrane-bound subunits. The data from simulations show rapid diffusion of water molecules in the protein interior, and formation of hydrated regions in the three antiporter-type subunits. An unexpected water-protein based connectivity between the middle of the Q-tunnel and the fourth proton channel is also observed. The protonation-state dependent dynamics of key acidic residues in the Nqo8 subunit suggest that the latter may be linked to redox-coupled proton pumping in complex I. We propose that in complex I the proton and electron transfer paths are not entirely separate, instead the nature of coupling may in part be 'direct'.