Browsing by Subject "OXIDOREDUCTASE"

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  • Gospodaryov, Dmytro V.; Lushchak, Oleh V.; Rovenko, Bohdana M.; Perkhulyn, Natalia V.; Gerards, Mike; Tuomela, Tea; Jacobs, Howard T. (2014)
  • Galemou Yoga, Etienne; Parey, Kristian; Djurabekova, Amina; Haapanen, Outi; Siegmund, Karin; Zwicker, Klaus; Sharma, Vivek; Zickermann, Volker; Angerer, Heike (2020)
    Respiratory complex I catalyzes electron transfer from NADH to ubiquinone (Q) coupled to vectorial proton translocation across the inner mitochondrial membrane. Despite recent progress in structure determination of this very large membrane protein complex, the coupling mechanism is a matter of ongoing debate and the function of accessory subunits surrounding the canonical core subunits is essentially unknown. Concerted rearrangements within a cluster of conserved loops of central subunits NDUFS2 (beta 1-beta 2(S2) loop), ND1 (TMH5-6(ND1) loop) and ND3 (TMH1-2(ND3) loop) were suggested to be critical for its proton pumping mechanism. Here, we show that stabilization of the TMH1-2(ND3) loop by accessory subunit LYRM6 (NDUFA6) is pivotal for energy conversion by mitochondrial complex I. We determined the high-resolution structure of inactive mutant F89A(LYRM6) of eukaryotic complex I from the yeast Yarrowia lipolytica and found long-range structural changes affecting the entire loop cluster. In atomistic molecular dynamics simulations of the mutant, we observed conformational transitions in the loop cluster that disrupted a putative pathway for delivery of substrate protons required in Q redox chemistry. Our results elucidate in detail the essential role of accessory subunit LYRM6 for the function of eukaryotic complex I and offer clues on its redox-linked proton pumping mechanism. Respiratory complex I plays a key role in energy metabolism. Cryo-EM structure of a mutant accessory subunit LYRM6 from the yeast Yarrowia lipolytica and molecular dynamics simulations reveal conformational changes at the interface between LYRM6 and subunit ND3, propagated further into the complex. These findings offer insight into the mechanism of proton pumping by respiratory complex I.
  • Kuleta, Patryk; Sarewicz, Marcin; Postila, Pekka; Rog, Tomasz; Osyczka, Artur (2016)
    Describing dynamics of proton transfers in proteins is challenging, but crucial for understanding processes which use them for biological functions. In cytochrome bc(1), one of the key enzymes of respiration or photosynthesis, proton transfers engage in oxidation of quinol (QH(2)) and reduction of quinone (Q) taking place at two distinct catalytic sites. Here we evaluated by site-directed mutagenesis the contribution of Lys251/Asp252 pair (bacterial numbering) in electron transfers and associated with it proton uptake to the quinone reduction site (Q(i) site). We showed that the absence of protonable group at position 251 or 252 significantly changes the equilibrium levels of electronic reactions including the Q(i)-site mediated oxidation of heme b(H), reverse reduction of heme b(H) by quinol and heme b(H)/Q(i) semiquinone equilibrium. This implicates the role of H-bonding network in binding of quinone/semiquinone and defining thermodynamic properties of Q/SQ/QH(2) triad. The Lys251/Asp252 proton path is disabled only when both protonable groups are removed. With just one protonable residue from this pair, the entrance of protons to the catalytic site is sustained, albeit at lower rates, indicating that protons can travel through parallel routes, possibly involving water molecules. This shows that proton paths display engineering tolerance for change as long as all the elements available for functional cooperation secure efficient proton delivery to the catalytic site. (C) 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license.
  • 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.
  • Uno, Shinpei; Masuya, Takahiro; Shinzawa-Itoh, Kyoko; Lasham, Jonathan; Haapanen, Outi; Shiba, Tomoo; Inaoka, Daniel Ken; Sharma, Vivek; Murai, Masatoshi; Miyoshi, Hideto (2020)
    NADH-quinone oxidoreductase (complex I) couples electron transfer from NADH to quinone with proton translocation across the membrane. Quinone reduction is a key step for energy transmission from the site of quinone reduction to the remotely located proton-pumping machinery of the enzyme. Although structural biology studies have proposed the existence of a long and narrow quinone-access channel, the physiological relevance of this channel remains debatable. We investigated here whether complex I in bovine heart submitochondrial particles (SMPs) can catalytically reduce a series of oversized ubiquinones (OS-UQs), which are highly unlikely to transit the narrow channel because their side chain includes a bulky ?block? that is ?13 ? across. We found that some OS-UQs function as efficient electron acceptors from complex I, accepting electrons with an efficiency comparable with ubiquinone-2. The catalytic reduction and proton translocation coupled with this reduction were completely inhibited by different quinone-site inhibitors, indicating that the reduction of OS-UQs takes place at the physiological reaction site for ubiquinone. Notably, the proton-translocating efficiencies of OS-UQs significantly varied depending on their side-chain structures, suggesting that the reaction characteristics of OS-UQs affect the predicted structural changes of the quinone reaction site required for triggering proton translocation. These results are difficult to reconcile with the current channel model; rather, the access path for ubiquinone may be open to allow OS-UQs to access the reaction site. Nevertheless, contrary to the observations in SMPs, OS-UQs were not catalytically reduced by isolated complex I reconstituted into liposomes. We discuss possible reasons for these contradictory results.