Browsing by Subject "Electron transfer"

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  • Haapanen, Outi; Sharma, Vivek (2018)
    Abstract Respiratory complex I is a giant redox-driven proton pump, and central to energy production in mitochondria and bacteria. It catalyses the reduction of quinone to quinol, and converts the free energy released into the endergonic proton translocation across the membrane. The proton pumping sets up the proton electrochemical gradient, which propels the synthesis of ATP. Despite the availability of extensive biochemical, biophysical and structural data on complex I, the mechanism of coupling between the electron and proton transfer reactions remain uncertain. In this work, we discuss current state-of-the-art in the field with particular emphasis on the molecular mechanism of respiratory complex I, as deduced from computational modeling and simulation approaches, but in strong alliance with the experimental data. This leads to novel synthesis of mechanistic ideas on a highly complex enzyme of the electron transport chain that has been associated with a number of mitochondrial and neurodegenerative disorders.
  • 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.
  • Haapanen, Outi; Sharma, Vivek (2021)
    Respiratory complex I is a key enzyme in the electron transport chains of mitochondria and bacteria. It transfers two electrons to quinone and couples this redox reaction to proton pumping to electrically charge the membrane it is embedded in. The charge and pH gradient across the membrane drives the synthesis of ATP. The redox reaction and proton pumping in complex I are separated in space and time, which raises the question of how the two reactions are coupled so efficiently. Here, we focus on the unique similar to 35 angstrom long tunnel of complex I, which houses the binding site of quinone reduction. We discuss the redox and protonation reactions that occur in this tunnel and how they influence the dynamics of protein and substrate. On the basis of recent structural data and results from molecular simulations, we review how quinone reduction and dynamics may be coupled to proton pumping in complex I.
  • Malkamäki, Aapo; Meunier, Brigitte; Reidelbach, Marco; Rich, Peter R.; Sharma, Vivek (2019)
    Cytochrome c oxidases (CcOs) in the respiratory chains of mitochondria and bacteria are primary consumers of molecular oxygen, converting it to water with the concomitant pumping of protons across the membrane to establish a proton electrochemical gradient. Despite a relatively well understood proton pumping mechanism of bacterial CcOs, the role of the H channel in mitochondrial forms of CcO remains debated. Here, we used site-directed mutagenesis to modify a central residue of the lower span of the H channel, Q413, in the genetically tractable yeast Saccharomyces cerevisiae. Exchange of Q413 to several different amino acids showed no effect on rates and efficiencies of respiratory cell growth, and redox potential measurements indicated minimal electrostatic interaction between the 413 locus and the nearest redox active component heme a. These findings clearly exclude a primary role of this section of the H channel in proton pumping in yeast CcO. In agreement with the experimental data, atomistic molecular dynamics simulations and continuum electrostatic calculations on wildtype and mutant yeast CcOs highlight potential bottlenecks in proton transfer through this route. Our data highlight the preference for neutral residues in the 413 locus, precluding sufficient hydration for formation of a proton conducting wire.
  • Sharma, Vivek; Wikstrom, Marten (2016)
    The active site of cytochrome c oxidase (CcO) comprises an oxygen-binding heme, a nearby copper ion (Cue), and a tyrosine residue that is covalently linked to one of the histidine ligands of Cu-B. Two proton-conducting pathways are observed in CcO, namely the D-and the K-channels, which are used to transfer protons either to the active site of oxygen reduction (substrate protons) or for pumping. Proton transfer through the D-channel is very fast, and its role in efficient transfer of both substrate and pumped protons is well established. However, it has not been fully clear why a separate K-channel is required, apparently for the supply of substrate protons only. In this work, we have analysed the available experimental and computational data, based on which we provide new perspectives on the role of the K-channel. Our analysis suggests that proton transfer in the K-channel may be gated by the protonation state of the active-site tyrosine (Tyr244) and that the neutral radical form of this residue has a more general role in the CcO mechanism than thought previously. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi. (C) 2016 Elsevier B.V. All rights reserved.