title: Mimicking respiratory phosphorylation using purified enzymes.
abstract: The enzymes of oxidative phosphorylation is a striking example of the functional association of multiple enzyme complexes, working together to form ATP from cellular reducing equivalents. These complexes, such as cytochrome c oxidase or the ATP synthase, are typically investigated individually and therefore, their functional interplay is not well understood. Here, we present methodology that allows the co-reconstitution of purified terminal oxidases and ATP synthases in synthetic liposomes. The enzymes are functionally coupled via proton translocation where upon addition of reducing equivalents the oxidase creates and maintains a transmembrane electrochemical proton gradient that energizes the synthesis of ATP by the F1F0 ATP synthase. The method has been tested with the ATP synthases from Escherichia coli and spinach chloroplasts, and with the quinol and cytochrome c oxidases from E. coli and Rhodobacter sphaeroides, respectively. Unlike in experiments with the ATP synthase reconstituted alone, the setup allows in vitro ATP synthesis under steady state conditions, with rates up to 90 ATP×s(-1)×enzyme(-1). We have also used the novel system to study the phenomenon of "mild uncoupling" as observed in mitochondria upon addition of low concentrations of ionophores (e.g. FCCP, SF6847) and the recoupling effect of 6-ketocholestanol. While we could reproduce the described effects, our data with the in vitro system does not support the idea of a direct interaction between a mitochondrial protein and the uncoupling agents as proposed earlier.
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland. Electronic address: christoph.vonballmoos@dcb.unibe.ch.
authors.forename: Christoph
authors.initials: C
authors.lastname: von Ballmoos
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland.
authors.forename: Olivier
authors.initials: O
authors.lastname: Biner
authors.affiliation: Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Tobias
authors.initials: T
authors.lastname: Nilsson
authors.affiliation: Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, SE-106 91 Stockholm, Sweden.
authors.forename: Peter
authors.initials: P
authors.lastname: Brzezinski
mesh_terms: D000255:Adenosine Triphosphate / Q000096:biosynthesis; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D003576:Electron Transport Complex IV / Q000378:metabolism*; D025261:Mitochondrial Proton-Translocating ATPases / Q000378:metabolism*; D018716:Molecular Mimicry; D010085:Oxidative Phosphorylation
chemical_list: D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D000255:Adenosine Triphosphate; D003576:Electron Transport Complex IV; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases
keywords: ATP synthesis; Ionophore; Lateral proton diffusion; Liposomes; Mild uncoupling; Respiratory chain