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

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.

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Boric Acid‐Fueled ATP Synthesis by FoF1 ATP Synthase Reconstituted in a Supramolecular Architecture

Angewandte Chemie ◽

10.1002/ange.202016253 ◽

2020 ◽

Author(s):

Junbai Li ◽

Xia Xu ◽

Jinbo Fei ◽

Youqian Xu ◽

Guangle Li ◽

...

Keyword(s):

Boric Acid ◽

Atp Synthase ◽

Atp Synthesis ◽

Supramolecular Architecture ◽

Fof1 Atp Synthase

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Mutation of a single residue in the ba3 oxidase specifically impairs protonation of the pump site

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1422434112 ◽

2015 ◽

Vol 112 (11) ◽

pp. 3397-3402 ◽

Cited By ~ 16

Author(s):

Christoph von Ballmoos ◽

Nathalie Gonska ◽

Peter Lachmann ◽

Robert B. Gennis ◽

Pia Ädelroth ◽

...

Keyword(s):

Electron Transfer ◽

Time Constant ◽

Thermus Thermophilus ◽

Proton Translocation ◽

Proton Pumping ◽

Wild Type ◽

Structural Variant ◽

In The Wild ◽

Membrane Bound ◽

Proton Uptake

The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 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 ba3 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 O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs 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 ∼1.2 ms was slowed 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 ba3 cytochrome c oxidase.

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Structure of a bacterial ATP synthase

10.1101/463968 ◽

2018 ◽

Cited By ~ 1

Author(s):

Hui Guo ◽

Toshiharu Suzuki ◽

John L. Rubinstein

Keyword(s):

Atp Synthase ◽

Biochemical Analysis ◽

Atp Hydrolysis ◽

Genetic Manipulation ◽

Proton Translocation ◽

Atp Synthesis ◽

Mitochondrial Complex ◽

Rotational States ◽

Membrane Region ◽

Atp Synthases

AbstractATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed theBacillusPS3 ATP synthase inEschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunitεshows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.

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Breaking the Carboxyl Rule

Journal of Biological Chemistry ◽

10.1074/jbc.m113.465138 ◽

2013 ◽

Vol 288 (29) ◽

pp. 21254-21265 ◽

Cited By ~ 20

Author(s):

Sergei P. Balashov ◽

Lada E. Petrovskaya ◽

Eleonora S. Imasheva ◽

Evgeniy P. Lukashev ◽

Andrei K. Dioumaev ◽

...

Keyword(s):

Schiff Base ◽

Water Molecule ◽

Proton Pump ◽

Rate Constants ◽

Proton Donor ◽

Carboxyl Group ◽

Amino Group ◽

Wild Type ◽

Fast Phase ◽

Proton Uptake

A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5 ± 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pKa of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ϵ-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.

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

Biochemical Journal ◽

10.1042/bj3460041 ◽

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.

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Structure and conformational states of the bovine mitochondrial ATP synthase by cryo-EM

eLife ◽

10.7554/elife.10180 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 180

Author(s):

Anna Zhou ◽

Alexis Rohou ◽

Daniel G Schep ◽

John V Bason ◽

Martin G Montgomery ◽

...

Keyword(s):

Atp Synthase ◽

Catalytic Mechanism ◽

Proton Translocation ◽

Atp Synthesis ◽

Bioinformatic Analysis ◽

Mitochondrial Atp Synthase ◽

A Subunit ◽

Classification Of Images ◽

Atp Synthases

Adenosine triphosphate (ATP), the chemical energy currency of biology, is synthesized in eukaryotic cells primarily by the mitochondrial ATP synthase. ATP synthases operate by a rotary catalytic mechanism where proton translocation through the membrane-inserted FO region is coupled to ATP synthesis in the catalytic F1 region via rotation of a central rotor subcomplex. We report here single particle electron cryomicroscopy (cryo-EM) analysis of the bovine mitochondrial ATP synthase. Combining cryo-EM data with bioinformatic analysis allowed us to determine the fold of the a subunit, suggesting a proton translocation path through the FO region that involves both the a and b subunits. 3D classification of images revealed seven distinct states of the enzyme that show different modes of bending and twisting in the intact ATP synthase. Rotational fluctuations of the c8-ring within the FO region support a Brownian ratchet mechanism for proton-translocation-driven rotation in ATP synthases.

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Perfect chemomechanical coupling of FoF1-ATP synthase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1700801114 ◽

2017 ◽

Vol 114 (19) ◽

pp. 4960-4965 ◽

Cited By ~ 25

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.

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