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  • 1. Gigliobianco, Tiziana
    et al.
    Gangolf, Marjorie
    Lakaye, Bernard
    Pirson, Bastien
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wins, Pierre
    Bettendorff, Lucien
    An alternative role of FoF1-ATP synthase in Escherichia coli: synthesis of thiamine triphosphate2013In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 3, p. 1071-Article in journal (Refereed)
    Abstract [en]

    In E. coli, thiamine triphosphate (ThTP), a putative signaling molecule, transiently accumulates in response to amino acid starvation. This accumulation requires the presence of an energy substrate yielding pyruvate. Here we show that in intact bacteria ThTP is synthesized from free thiamine diphosphate (ThDP) and P-i, the reaction being energized by the proton-motive force (Delta p) generated by the respiratory chain. ThTP production is suppressed in strains carrying mutations in F-1 or a deletion of the atp operon. Transformation with a plasmid encoding the whole atp operon fully restored ThTP production, highlighting the requirement for FoF1-ATP synthase in ThTP synthesis. Our results show that, under specific conditions of nutritional downshift, FoF1-ATP synthase catalyzes the synthesis of ThTP, rather than ATP, through a highly regulated process requiring pyruvate oxidation. Moreover, this chemiosmotic mechanism for ThTP production is conserved from E. coli to mammalian brain mitochondria.

  • 2. Lee, Chiara
    et al.
    Kang, Hae Joo
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Newstead, Simon
    Uzdavinys, Povilas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dotson, David L.
    Iwata, So
    Beckstein, Oliver
    Cameron, Alexander D.
    Drew, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Imperial College London .
    A two-domain elevator mechanism for sodium/proton antiport2013In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 501, no 7468, p. 573-577Article in journal (Refereed)
    Abstract [en]

    Sodium/proton (Na+/H+) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis1. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets(2). The best understood model system for Na+/H+ antiport is NhaA from Escherichia coli(1,3), for which both electron microscopy and crystal structures are available(4-6). NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein(1,4). Likemany Na+/H+ antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur(7). The only reported NhaA crystal structure so far is of the low pH inactivated form(4). Here we describe the active-state structure of a Na+/H+ antiporter, NapA from Thermus thermophilus, at 3 angstrom resolution, solved from crystals grown at pH7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding(1,8,9) directly, a role supported hereby molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 20 degrees against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second(3), Na+/H+ antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.

  • 3. Lee, Chiara
    et al.
    Yashiro, Shoko
    Dotson, David L.
    Uzdavinys, Povilas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Iwata, So
    Sansom, Mark S. P.
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Beckstein, Oliver
    Drew, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Imperial College London, England.
    Cameron, Alexander D.
    Crystal structure of the sodium-proton antiporter NhaA dimer and new mechanistic insights2014In: The Journal of General Physiology, ISSN 0022-1295, E-ISSN 1540-7748, Vol. 144, no 6, p. 529-544Article in journal (Refereed)
    Abstract [en]

    Sodium-proton antiporters rapidly exchange protons and sodium ions across the membrane to regulate intracellular pH, cell volume, and sodium concentration. How ion binding and release is coupled to the conformational changes associated with transport is not clear. Here, we report a crystal form of the prototypical sodium-proton antiporter NhaA from Escherichia coli in which the protein is seen as a dimer. In this new structure, we observe a salt bridge between an essential aspartic acid (Asp163) and a conserved lysine (Lys300). An equivalent salt bridge is present in the homologous transporter NapA, but not in the only other known crystal structure of NhaA, which provides the foundation of most existing structural models of electrogenic sodium-proton antiport. Molecular dynamics simulations show that the stability of the salt bridge is weakened by sodium ions binding to Asp164 and the neighboring Asp163. This suggests that the transport mechanism involves Asp163 switching between forming a salt bridge with Lys300 and interacting with the sodium ion. pK(a) calculations suggest that Asp163 is highly unlikely to be protonated when involved in the salt bridge. As it has been previously suggested that Asp163 is one of the two residues through which proton transport occurs, these results have clear implications to the current mechanistic models of sodium-proton antiport in NhaA.

  • 4.
    Lindholm, Ljubica
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ariöz, Candan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Jawurek, Michael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Liebau, Jobst
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mäler, Lena
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Barth, Andreas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Effect of lipid bilayer properties on the photocycle of green proteorhodopsin2015In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1847, no 8, p. 698-708Article in journal (Refereed)
    Abstract [en]

    The significance of specific lipids for proton pumping by the bacterial rhodopsin proteorhodopsin (pR) was studied. To this end, it was examined whether pR preferentially binds certain lipids and whether molecular properties of the lipid environment affect the photocycle. pR's photocyde was followed by microsecond flash-photolysis in the visible spectral range. It was fastest in phosphatidylcholine liposomes (soy bean lipid), intermediate in 3-[(3-cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS): 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bicelles and in Triton X-100, and slowest when pR was solubilized in CHAPS. In bicelles with different lipid compositions, the nature of the head groups, the unsaturation level and the fatty acid chain length had small effects on the photocycle. The specific affinity of pR for lipids of the expression host Eschetichia coil was investigated by an optimized method of lipid isolation from purified membrane protein using two different concentrations of the detergent N-dodecyl-beta-D-maltoside (DDM). We found that 11 lipids were copurified per pR molecule at 0.1% DDM, whereas essentially all lipids were stripped off from pR by 1% DDM. The relative amounts of copurifled phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin did not correlate with the molar percentages normally present in E. coil cells. The results indicate a predominance of phosphatidylethanolamine species in the lipid annulus around recombinant pR that are less polar than the dominant species in the cell membrane of the expression host E. coli.

  • 5.
    Lundgren, Camilla A. K
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sjöstrand, Dan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Biner, Olivier
    Bennett, Matthew
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rudling, Axel
    Johansson, Ann-Louise
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinsk, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Carlsson, Jens
    von Ballmoos, Christoph
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Scavenging of superoxide by a membrane-bound superoxide oxidase2018In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 14, p. 788-793Article in journal (Refereed)
    Abstract [en]

    Superoxide is a reactive oxygen species produced during aerobic metabolism in mitochondria and prokaryotes. It causes damage to lipids, proteins and DNA and is implicated in cancer, cardiovascular disease, neurodegenerative disorders and aging. As protection, cells express soluble superoxide dismutases, disproportionating superoxide to oxygen and hydrogen peroxide. Here, we describe a membrane-bound enzyme that directly oxidizes superoxide and funnels the sequestered electrons to ubiquinone in a diffusion-limited reaction. Experiments in proteoliposomes and inverted membranes show that the protein is capable of efficiently quenching superoxide generated at the membrane in vitro. The 2.0 Å crystal structure shows an integral membrane di-heme cytochrome b poised for electron transfer from the P-side and proton uptake from the N-side. This suggests that the reaction is electrogenic and contributes to the membrane potential while also conserving energy by reducing the quinone pool. Based on this enzymatic activity, we propose that the enzyme family be denoted superoxide oxidase (SOO).

  • 6.
    Nilsson, Tobias
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rydström Lundin, Camilla
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordlund, Gustav
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Bern, Switzerland.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lipid-mediated Protein-protein Interactions Modulate Respiration-driven ATP Synthesis2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 24113Article in journal (Refereed)
    Abstract [en]

    Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane which used, e.g. for generation of ATP by the ATP synthase. Here, we have co-reconstituted the proton pump cytochrome bo3 (ubiquinol oxidase) together with ATP synthase in liposomes and studied the effect of changing the lipid composition on the ATP synthesis activity driven by proton pumping. We found that for 100 nm liposomes, containing 5 of each proteins, the ATP synthesis rates decreased significantly with increasing fractions of DOPA, DOPE, DOPG or cardiolipin added to liposomes made of DOPC; with e.g. 5% DOPG, we observed an almost 50% decrease in the ATP synthesis rate. However, upon increasing the average distance between the proton pumps and ATP synthases, the ATP synthesis rate dropped and the lipid dependence of this activity vanished. The data indicate that protons are transferred along the membrane, between cytochrome bo3 and the ATP synthase, but only at sufficiently high protein densities. We also argue that the local protein density may be modulated by lipid-dependent changes in interactions between the two proteins complexes, which points to a mechanism by which the cell may regulate the overall activity of the respiratory chain.

  • 7.
    Nordlund, Gustav
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    SNARE-fusion mediated insertion of membrane proteins into native and artificial membranesManuscript (preprint) (Other academic)
  • 8.
    Nordlund, Gustav
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Bern, Switzerland.
    SNARE-fusion mediated insertion of membrane proteins into native and artificial membranes2014In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 5, p. 4303-Article in journal (Refereed)
    Abstract [en]

    Membrane proteins carry out functions such as nutrient uptake, ATP synthesis or transmembrane signal transduction. An increasing number of reports indicate that cellular processes are underpinned by regulated interactions between these proteins. Consequently, functional studies of these networks at a molecular level require co-reconstitution of the interacting components. Here, we report a SNARE protein-based method for incorporation of multiple membrane proteins into artificial membrane vesicles of well-defined composition, and for delivery of large water-soluble substrates into these vesicles. The approach is used for in vitro reconstruction of a fully functional bacterial respiratory chain from purified components. Furthermore, the method is used for functional incorporation of the entire F1F0 ATP synthase complex into native bacterial membranes from which this component had been genetically removed. The novel methodology offers a tool to investigate complex interaction networks between membrane-bound proteins at a molecular level, which is expected to generate functional insights into key cellular functions.

  • 9.
    Nordlund, Gustav
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rydström Lundin, Camilla
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, Tobias
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Effect of lipid composition on respiration-driven ATP synthesisManuscript (preprint) (Other academic)
    Abstract [en]

    Energy conversion in biological systems is underpinned by membrane-bound proton transporters that generate and maintain a proton electrochemical gradient across the membrane. The free energy stored in this gradient is used, for example, for transport of molecules or ions by other transporters as well as for generation of ATP by the ATP synthase. Understanding the overall process requires functional studies of the coupled reactions, which involve co-reconstitution of e.g. a proton pump and a transporter that utilizes the proton gradient. This process is likely to be further influenced by the composition of the membrane, which, for example, may facilitate lateral proton transfer. In the present study, we have co-reconstituted the proton pump bo3 ubiquinol oxidase with ATP synthase, both from E. coli, in liposomes with different membrane compositions. The coupled proton pumping and ATP-synthesis activities were investigated. We found that the ATP synthesis was significantly higher in a membrane composed of pure DOPC lipids than in the presence of DOPA, DOPE, DOPG or cardiolipin. The drop in activity was considerably more pronounced upon addition of the negatively charged head groups (PA, PG or cardiolipin) than upon addition of the zwitterionic PE. The origin of these effects is discussed.

  • 10.
    Näsvik Öjemyr, Linda
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Faxén, Kristina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Svahn, Emelie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The membrane modulates internal proton transfer in cytochrome c oxidase2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, p. 1092-1100Article in journal (Refereed)
    Abstract [en]

    The functionality of membrane proteins is often modulated by the surrounding membrane. Here, we investigated the effect of membrane reconstitution of purified cytochrome c oxidase (CytcO) on the kinetics and thermodynamics of internal electron and proton-transfer reactions during O2 reduction. Reconstitution of the detergent-solubilized enzyme in small unilamellar soybean phosphatidylcholine vesicles resulted in a lowering of the pKa in the pH dependence profile of the proton-uptake rate. This pKa change resulted in decreased proton-uptake rates in the pH range of 6.5–9.5, which is explained in terms of lowering of the pKa of an internal proton donor within CytcO. At pH 7.5, the rate decreased to the same extent when vesicles were prepared from the pure zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or the anionic lipid 1,2-dioleoyl-sn-glycero-3-phospho(1-rac-glycerol) (DOPG). In addition, a small change in the internal CuA–heme a electron equilibrium constant was observed. This effect was lipid-dependent and explained in terms of a lower electrostatic potential within the membrane-spanning part of the protein with the anionic DOPG lipids than with the zwitterionic DOPC lipids. In conclusion, the data show that the membrane significantly modulates internal charge-transfer reactions and thereby the function of the membrane-bound enzyme.

  • 11.
    Näsvik Öjemyr, Linda
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gennis, Robert B.
    Department of Biochemistry, University of Illinois at Urbana-Champaign.
    Sligar, Stephen G.
    Department of Chemistry, University of Illinois at Urbana-Champaign.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reconstitution of respiratory oxidases in membrane nanodiscs for investigation of proton-coupled electron transfer2012In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 586, p. 640-645Article in journal (Refereed)
    Abstract [en]

    The function of membrane-bound transporters is commonly affected by the milieu of the hydrophobic, membrane-spanning part of the transmembrane protein. Consequently, functional studies of these proteins often involve incorporation into a native-like bilayer where the lipid components of the membrane can be controlled. The classical approach is to reconstitute the purified protein into liposomes. Even though the use of such liposomes is essential for studies of transmembrane transport processes in general, functional studies of the transporters themselves in liposomes suffer from several disadvantages. For example, transmembrane proteins can adopt two different orientations when reconstituted into liposomes, and one of these populations may be inaccessible to ligands, to changes in pH or ion concentration in the external solution. Furthermore, optical studies of proteins reconstituted in liposomes suffer from significant light scattering, which diminishes the signal-to-noise value of the measurements. One attractive approach to circumvent these problems is to use nanodiscs, which are phospholipid bilayers encircled by a stabilizing amphipathic helical membrane scaffold protein. These membrane nanodiscs are stable, soluble in aqueous solution without detergent and do not scatter light significantly. In the present study, we have developed a protocol for reconstitution of the aa3- and ba3-type cytochrome c oxidases into nanodiscs. Furthermore, we studied proton-coupled electron-transfer reactions in these enzymes with microsecond time resolution. The data show that the nanodisc membrane environment accelerates proton uptake in both oxidases.

  • 12.
    Oliynyk, Vitaliy
    et al.
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Mille, Christian
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK), Materials Chemistry.
    Ng, Jovice B. S.
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Corkery, Robert W.
    Bergström, Lennart
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK), Materials Chemistry.
    Selective and atp driven transport of ions across supported membranes into nanoporous carriers using gramicidin a and atp synthase2013In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 15, no 8, p. 2733-2740Article in journal (Refereed)
    Abstract [en]

    We report a robust and versatile membrane protein based system for selective uptake and release of ions from nanoporous particles sealed with ion-tight lipid bilayers of various compositions that is driven by the addition of ATP or a chemical potential gradient. We have successfully incorporated both a passive ion channel-type peptide (gramicidin A) and a more complex primary sodium ion transporter (ATP synthase) into the supported lipid bilayers on solid nanoporous silica particles. Protein-mediated controlled release/uptake of sodium ions across the ion-tight lipid bilayer seal from or into the nanoporous silica carrier was imaged in real time using a confocal laser scanning microscope and the intensity changes were quantified. ATP-driven transport of sodium ions across the supported lipid bilayer against a chemical gradient was demonstrated. The possibility of designing durable carriers with tight lipid membranes, containing membrane proteins for selective ion uptake and release, offers new possibilities for functional studies of single or cascading membrane protein systems and could also be used as biomimetic microreactors for controlled synthesis of inorganic multicomponent materials.

  • 13. Oliynyk, Vitaliy
    et al.
    Mille, Christian
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Ng, Jovice B S
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Corkery, Robert W
    Bergström, Lennart
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Selective and ATP-driven transport of ions across supported membranes into nanoporous carriers using gramicidin A and ATP synthaseArticle in journal (Refereed)
  • 14.
    Smirnova, Irina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Chang, Hsin-Yang
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Adelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gennis, Robert B.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Single Mutations That Redirect Internal Proton Transfer in the ba(3) Oxidase from Thermus thermophilus2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 40, p. 7022-7030Article in journal (Refereed)
    Abstract [en]

    The ba(3)-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound proton pump. Results from earlier studies have shown that with the aa(3)-type oxidases proton uptake to the catalytic site and pump site occurs simultaneously. However, with ba(3) oxidase the pump site is loaded before proton transfer to the catalytic site because the proton transfer to the latter is slower than that with the aa(3) oxidases. In addition, the timing of formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, we have investigated two mutant ba(3) CytcOs in which residues of the proton pathway leading to the catalytic site as well as the pump site were exchanged, Thr312Val and Tyr244Phe. Even though ba(3) CytcO uses only a single proton pathway for transfer of the substrate and pumped protons, the amino-acid residue substitutions had distinctly different effects on the kinetics of proton transfer to the catalytic site and the pump site. The results indicate that the rates of these reactions can be modified independently by replacement of single residues within the proton pathway. Furthermore, the data suggest that the Thr312Val and Tyr244Phe mutations interfere with a structural rearrangement in the proton pathway that is rate limiting for proton transfer to the catalytic site.

  • 15.
    Smirnova, Irina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Reimann, Joachim
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Chang, Hsin-Yang
    Gennis, Robert B.
    Fee, James A.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Functional Role of Thr-312 and Thr-315 in the Proton-Transfer Pathway in ba3 Cytochrome c Oxidase from Thermus thermophilus2010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 33, p. 7033-7039Article in journal (Refereed)
    Abstract [en]

    Cytochrome ba3 from Thermus thermophilus is a member of the family of B-type heme-copper oxidases, which have a low degree of sequence homology to the well-studied mitochondrial-like A-type enzymes. Recently, it was suggested that the ba3 oxidase has only one pathway for the delivery of protons to the active site and that this pathway is spatially analogous to the K-pathway in the A-type oxidases [Chang, H.-Y., et al. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 16169−16173]. This suggested pathway includes two threonines at positions 312 and 315. In this study, we investigated the time-resolved reaction between fully reduced cytochrome ba3 and O2 in variants where Thr-312 and Thr-315 were modified. While in the A-type oxidases this reaction is essentially unchanged in variants with the K-pathway modified, in the Thr-312 → Ser variant in the ba3 oxidase both reactions associated with proton uptake from solution, the PR → F and F → O transitions, were slowed compared to those of wild-type ba3. The observed time constants were slowed 3-fold (for PR → F, from 60 to 170 μs in the wild type) and 30-fold (for F → O, from 1.1 to 40 ms). In the Thr-315 → Val variant, the F → O transition was 5-fold slower (5 ms) than for the wild-type oxidase, whereas the PR → F transition displayed an essentially unchanged time constant. However, the uptake of protons from solution was a factor of 2 slower and decoupled from the optical PR → F transition. Our results thus show that proton uptake is significantly and specifically inhibited in the two variants, strongly supporting the suggested involvement of T312 and T315 in the transfer of protons to the active site during O2 reduction in the ba3 oxidase.

  • 16.
    von Ballmoos, Christoph
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Adelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gennis, Robert B.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Proton transfer in ba(3) cytochrome c oxidase from Thermus thermophilus2012In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1817, no 4, p. 650-657Article, review/survey (Refereed)
    Abstract [en]

    The respiratory heme-copper oxidases catalyze reduction of O-2 to H2O, linking this process to transmembrane proton pumping. These oxidases have been classified according to the architecture, location and number of proton pathways. Most structural and functional studies to date have been performed on the A-class oxidases, which includes those that are found in the inner mitochondrial membrane and bacteria such as Rhodobacter sphaeroides and Paracoccus denitrificans (aa(3)-type oxidases in these bacteria). These oxidases pump protons with a stoichiometry of one proton per electron transferred to the,catalytic site. The bacterial A-class oxidases use two proton pathways (denoted by letters D and K, respectively), for the transfer of protons to the catalytic site, and protons that are pumped across the membrane. The B-type oxidases such as, for example, the ba(3) oxidase from Thermus thermophilus, pump protons with a lower stoichiometry of 0.5 H+/electron and use only one proton pathway for the transfer of all protons. This pathway overlaps in space with the K pathway in the A class oxidases without showing any sequence homology though. Here, we review the functional properties of the A- and the B-class ba3 oxidases with a focus on mechanisms of proton transfer and pumping. This article is part of a Special Issue entitled: Respiratory Oxidases.

  • 17.
    von Ballmoos, Christoph
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gennis, Robert B.
    Aedelroth, Pia
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kinetic design of the respiratory oxidases2011In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 27, p. 11057-11062Article in journal (Refereed)
    Abstract [en]

    Energy conservation in all kingdoms of life involves electron transfer, through a number of membrane-bound proteins, associated with proton transfer across the membrane. In aerobic organisms, the last component of this electron-transfer chain is a respiratory heme-copper oxidase that catalyzes reduction of O(2) to H(2)O, linking this process to transmembrane proton pumping. So far, the molecular mechanism of proton pumping is not known for any system that is driven by electron transfer. Here, we show that this problem can be addressed and elucidated in a unique cytochrome c oxidase (cytochrome ba(3)) from a thermophilic bacterium, Thermus thermophilus. The results show that in this oxidase the electron-and proton-transfer reactions are orchestrated in time such that previously unresolved proton-transfer reactions could be directly observed. On the basis of these data we propose that loading of the proton pump occurs upon electron transfer, but before substrate proton transfer, to the catalytic site. Furthermore, the results suggest that the pump site alternates between a protonated and deprotonated state for every second electron transferred to the catalytic site, which would explain the noninteger pumping stoichiometry (0.5 H(+)/e(-)) of the ba(3) oxidase. Our studies of this variant of Nature's palette of mechanistic solutions to a basic problem offer a route toward understanding energy conservation in biological systems.

  • 18.
    von Ballmoos, Christoph
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gonska, Nathalie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lachmann, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gennis, Robert B.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Mutation of a single residue in the ba(3) oxidase specifically impairs protonation of the pump site2015In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 11, p. 3397-3402Article in journal (Refereed)
    Abstract [en]

    The ba(3)-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O-2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba(3) oxidase where a putative pump site was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O-2 reduction. The results from our studies show that proton uptake to the pump site (time constant similar to 65 mu s in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of similar to 1.2 ms was slowed to similar to 8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba(3) cytochrome c oxidase.

  • 19.
    von Ballmoos, Christoph
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Lachmann, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gennis, Robert B.
    Adelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Timing of Electron and Proton Transfer in the ba(3) Cytochrome c Oxidase from Thermus thermophilus2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 22, p. 4507-4517Article in journal (Refereed)
    Abstract [en]

    Heme-copper oxidases are membrane-bound proteins that catalyze the reduction of O-2 to H2O, a highly exergonic reaction. Part of the free energy of this reaction is used for pumping of protons across the membrane. The ba(3) oxidase from Thermus thermophilus presumably uses a single proton pathway for the transfer of substrate protons used during O-2 reduction as well as for the transfer of the protons that are pumped across the membrane. The pumping stoichiometry (0.5 H+/electron) is lower than that of most other (mitochondrial-like) oxidases characterized to date (1 H+/electron). We studied the pH dependence and deuterium isotope effect of the kinetics of electron and proton transfer reactions in the ba3 oxidase. The results from these studies suggest that the movement of protons to the catalytic site and movement to a site located some distance from the catalytic site [proposed to be a proton-loading site (PLS) for pumped protons] are separated in time, which allows individual investigation of these reactions. A scenario in which the uptake and release of a pumped proton occurs upon every second transfer of an electron to the catalytic site would explain the decreased proton pumping stoichiometry compared to that of mitochondrial-like oxidases.

  • 20.
    von Ballmoos, Christoph
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Bern, Switzerland.
    Smirnova, Irina
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Poiana, Federica
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gonska, Nathalie
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Chang, Hsin-Yang
    Gennis, Robert B.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ädelroth, Pia
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dynamics of the K-B Proton Pathway in Cytochrome ba(3) from Thermus thermophilus2017In: Israel Journal of Chemistry, ISSN 0021-2148, Vol. 57, no 5, p. 424-436Article in journal (Refereed)
    Abstract [en]

    The ba(3) cytochrome c oxidase from Thermus thermophilus is a B-type oxygen-reducing heme-copper oxidase and a proton pump. It uses only one proton pathway for transfer of protons to the catalytic site, the K-B pathway. It was previously shown that the ba(3) oxidase has an overall similar reaction sequence to that in mitochondrial-like A-type oxidases. However, the timing of loading the pump site, and formation and decay of catalytic intermediates is different in the two types of oxidases. In the present study, we have investigated variants in which two amino acids of the K-B proton pathway leading to the catalytic site were exchanged; Tyr-248 (located approximate to 23 angstrom below the active site towards the cytoplasm) in subunit I (Y248T) and Glu-15 (approximate to 26 angstrom below the active site, approximate to 16 angstrom from Tyr-248) in subunit II (E15(II)Q). Even though the overall catalytic turnover in these two variants is similar and very low (<1% of wildtype), the substitutions had distinctly different effects on the kinetics of proton transfer to the catalytic site. The results indicate that the Glu-15(II) is the only essentially crucial residue of the K-B pathway, but that the Tyr-248 also plays a distinct role in defining an internal proton donor and controlling the dynamics of proton transfer to the pump site and the catalytic site.

  • 21.
    von Ballmoos, Christoph
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wiedenmann, Alexander
    Dimroth, Peter
    Essentials for ATP Synthesis by F1F0 ATP Synthases2009In: Annual Review of Biochemistry, ISSN 0066-4154, E-ISSN 1545-4509, Vol. 78, p. 649-672Article, review/survey (Refereed)
    Abstract [en]

    The majority of cellular energy in the form of adenosine triphosphate (ATP) is synthesized by the ubiquitous F1F0 ATP synthase. Power for ATP synthesis derives from an electrochemical proton (or Na+) gradient, which drives rotation of membranous F-0 motor components. Efficient rotation not only requires a significant driving force (Delta mu H+), consisting of membrane potential (Delta psi) and proton concentration gradient (Delta pH), but also a high proton concentration at the source P side. In vivo this is maintained by dynamic proton movements across and along the surface of the membrane. The torque-generating unit consists of the interface of the rotating c ring and the stator a subunit. Ion translocation through this unit involves a sophisticated interplay between the c-ring binding sites, the stator arginine, and the coupling ions on both sides of the membrane. c-ring rotation is transmitted to the eccentric shaft gamma-subunit to elicit conformational changes in the catalytic sites of F-1, leading to ATP synthesis.

  • 22. Wiedenmann, Alexander
    et al.
    Dimroth, Peter
    von Ballmoos, Christoph
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Functional asymmetry of the F-0 motor in bacterial ATP synthases2009In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 72, no 2, p. 479-490Article in journal (Refereed)
    Abstract [en]

    F1F0 ATP synthases use the electrochemical potential of H+ or Na+ across biological membranes to synthesize ATP by a rotary mechanism. In bacteria, the enzymes can act in reverse as ATP-driven ion pumps creating the indispensable membrane potential. Here, we demonstrate that the F-0 parts of a Na+- and H+-dependent enzyme display major asymmetries with respect to their mode of operation, reflected by the requirement of similar to 100 times higher Na+ or H+ concentrations for the synthesis compared with the hydrolysis of ATP. A similar asymmetry is observed during ion transport through isolated F-0 parts, indicating different affinities for the binding sites in the a/c interface. Together with further data, we propose a model that provides a rationale for a differential usage of membrane potential and ion gradient during ATP synthesis as observed experimentally. The functional asymmetry might also reflect an important property of the ATP synthesis mechanism in vivo. In Escherichia coli, we observed respiratory chain-driven ATP production at pH 7-8, while P-site pH values < 6.5 were required for ATP synthesis in vitro. This discrepancy is discussed with respect to the hypothesis that during respiration lateral proton diffusion could lead to significant acidification at the membrane surface.

1 - 22 of 22
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