scholarly journals Perfect chemomechanical coupling of FoF1-ATP synthase

2017 ◽  
Vol 114 (19) ◽  
pp. 4960-4965 ◽  
Author(s):  
Naoki Soga ◽  
Kazuya Kimura ◽  
Kazuhiko Kinosita ◽  
Masasuke Yoshida ◽  
Toshiharu Suzuki
Keyword(s):  
Atp Synthase ◽  
Chemical Potential ◽  
Initial Rate ◽  
Coupling Efficiency ◽  
Atp Synthesis ◽  
Energy Yield ◽  
Coupling Mechanism ◽  
Chemomechanical Coupling ◽  
Base Transition ◽  
Fof1 Atp Synthase

FoF1-ATP synthase (FoF1) couples H+ flow in Fo domain and ATP synthesis/hydrolysis in F1 domain through rotation of the central rotor shaft, and the H+/ATP ratio is crucial to understand the coupling mechanism and energy yield in cells. Although H+/ATP ratio of the perfectly coupling enzyme can be predicted from the copy number of catalytic β subunits and that of H+ binding c subunits as c/β, the actual H+/ATP ratio can vary depending on coupling efficiency. Here, we report actual H+/ATP ratio of thermophilic Bacillus FoF1, whose c/β is 10/3. Proteoliposomes reconstituted with the FoF1 were energized with ΔpH and Δψ by the acid−base transition and by valinomycin-mediated diffusion potential of K+ under various [ATP]/([ADP]⋅[Pi]) conditions, and the initial rate of ATP synthesis/hydrolysis was measured. Analyses of thermodynamically equilibrated states, where net ATP synthesis/hydrolysis is zero, show linear correlation between the chemical potential of ATP synthesis/hydrolysis and the proton motive force, giving the slope of the linear function, that is, H+/ATP ratio, 3.3 ± 0.1. This value agrees well with the c/β ratio. Thus, chemomechanical coupling between Fo and F1 is perfect.

scholarly journals ATP synthase K+- and H+-fluxes drive ATP synthesis and enable mitochondrial K+-‘uniporter’ function: I. Characterization of ion fluxes

Function ◽  
2021 ◽  
Author(s):  
Magdalena Juhaszova ◽  
Evgeny Kobrinsky ◽  
Dmitry B Zorov ◽  
H Bradley Nuss ◽  
Yael Yaniv ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Single Molecule ◽  
Chemical Potential ◽  
Atp Synthesis ◽  
Energy Utilization ◽  
Photon Detection ◽  
Isolated Mitochondria ◽  
Intact Mitochondrion ◽  
Atp Generation ◽  
K Transport

Abstract ATP synthase (F1Fo) synthesizes daily our body's weight in ATP, whose production-rate can be transiently increased several-fold to meet changes in energy utilization. Using purified mammalian F1Fo-reconstituted proteoliposomes and isolated mitochondria, we show F1Fo can utilize both ΔΨm-driven H+- and K+-transport to synthesize ATP under physiological pH = 7.2 and K+ = 140 mEq/L conditions. Purely K+-driven ATP synthesis from single F1Fo molecules measured by bioluminescence photon detection could be directly demonstrated along with simultaneous measurements of unitary K+ currents by voltage clamp, both blocked by specific Fo inhibitors. In the presence of K+, compared to osmotically-matched conditions in which this cation is absent, isolated mitochondria display 3.5-fold higher rates of ATP synthesis, at the expense of 2.6-fold higher rates of oxygen consumption, these fluxes being driven by a 2.7:1 K+:H+ stoichiometry. The excellent agreement between the functional data obtained from purified F1Fo single molecule experiments and ATP synthase studied in the intact mitochondrion under unaltered OxPhos coupling by K+ presence, is entirely consistent with K+ transport through the ATP synthase driving the observed increase in ATP synthesis. Thus, both K+ (harnessing ΔΨm) and H+ (harnessing its chemical potential energy, ΔµH) drive ATP generation during normal physiology.

scholarly journals Dye-ligand chromatographic purification of intact multisubunit membrane protein complexes: application to the chloroplast H+-F0F1-ATP synthase

2000 ◽  
Vol 346 (1) ◽  
pp. 41-44
Author(s):  
Holger SEELERT ◽  
Ansgar POETSCH ◽  
Meino ROHLFS ◽  
Norbert A. DENCHER
Keyword(s):  
Membrane Protein ◽  
Phosphatidic Acid ◽  
Ammonium Sulphate ◽  
Atp Synthase ◽  
Protein Complex ◽  
Protein Complexes ◽  
Atp Synthesis ◽  
Chromatographic Purification ◽  
Membrane Protein Complexes ◽  
Fof1 Atp Synthase

n-Dodecyl-β-D-maltoside was used as a detergent to solubilize the ammonium sulphate precipitate of chloroplast FOF1-ATP synthase, which was purified further by dye-ligand chromatography. Upon reconstitution of the purified protein complex into phosphatidylcholine/phosphatidic acid liposomes, ATP synthesis, driven by an artificial ∆pH/∆ψ, was observed. The highest activity was achieved with ATP synthase solubilized in n-dodecyl-β-D-maltoside followed by chromatography with Red 120 dye. The optimal dye for purification with CHAPS was Green 5. All known subunits were present in the monodisperse proton-translocating ATP synthase preparation obtained from chloroplasts.

scholarly journals The phototroph-specific β-hairpin structure of the γ subunit of FoF1-ATP synthase is important for efficient ATP-synthesis of cyanobacteria

2021 ◽  
pp. 101027
Author(s):  
Kumiko Kondo ◽  
Masayuki Izumi ◽  
Kosuke Inabe ◽  
Keisuke Yoshida ◽  
Mari Imashimizu ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Atp Synthesis ◽  
Hairpin Structure ◽  
Γ Subunit ◽  
Fof1 Atp Synthase

scholarly journals Cooperation among c-subunits of FoF1-ATP synthase in rotation-coupled proton translocation

2021 ◽  
Author(s):  
Noriyo Mitome ◽  
Shintaroh Kubo ◽  
Sumie Ohta ◽  
Hikaru Takashima ◽  
Yuto Shigefuji ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Proton Pump ◽  
Proton Translocation ◽  
Atp Synthesis ◽  
Wild Type ◽  
Single Chain ◽  
Proton Release ◽  
Biochemical Assays ◽  
Proton Uptake ◽  
Fof1 Atp Synthase

In FoF1-ATP synthase, proton translocation through Fo drives rotation of the c-subunit oligomeric ring relative to the a-subunit. Recent studies suggest that in each step of the rotation, key glutamic acid residues in different c-subunits contribute to proton release to and proton uptake from the a-subunit. However, no studies have demonstrated cooperativity among c-subunits toward FoF1-ATP synthase activity. Here, we addressed this using Bacillus PS3 ATP synthase harboring c-ring with various combinations of wild-type and cE56D, enabled by genetically fused single-chain c-ring. ATP synthesis and proton pump activities were significantly decreased by a single cE56D mutation and further decreased by double cE56D mutations. Moreover, activity further decreased as the two mutation sites were separated, indicating cooperation among c-subunits. Similar results were obtained for proton transfer-coupled molecular simulations. Simulations revealed that prolonged proton uptake in mutated c-subunits is shared between two c-subunits, explaining the cooperation observed in biochemical assays.

Assembly of the Escherichia coli FoF1 ATP synthase involves distinct subcomplex formation

2013 ◽  
Vol 41 (5) ◽  
pp. 1288-1293 ◽  
Author(s):  
Gabriele Deckers-Hebestreit
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Cytoplasmic Membrane ◽  
Atp Synthesis ◽  
E Coli ◽  
Assembly Pathway ◽  
Polypeptide Chains ◽  
Fof1 Atp Synthase

The ATP synthase (FoF1) of Escherichia coli couples the translocation of protons across the cytoplasmic membrane by Fo to ATP synthesis or hydrolysis in F1. Whereas good knowledge of the nanostructure and the rotary mechanism of the ATP synthase is at hand, the assembly pathway of the 22 polypeptide chains present in a stoichiometry of ab2c10α3β3γδϵ has so far not received sufficient attention. In our studies, mutants that synthesize different sets of FoF1 subunits allowed the characterization of individually formed stable subcomplexes. Furthermore, the development of a time-delayed in vivo assembly system enabled the subsequent synthesis of particular missing subunits to allow the formation of functional ATP synthase complexes. These observations form the basis for a model that describes the assembly pathway of the E. coli ATP synthase from pre-formed subcomplexes, thereby avoiding membrane proton permeability by a concomitant assembly of the open H+-translocating unit within a coupled FoF1 complex.

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