scholarly journals pH-dependent 11° F1FO ATP synthase sub-steps reveal insight into the FO torque generating mechanism

2021 ◽  
Author(s):  
Wayne D Frasch ◽  
Seiga Yanagisawa
Keyword(s):  
Atp Synthase ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Proton Pumping ◽  
Subunit A ◽  
Anticlockwise Rotation ◽  
C Subunit ◽  
Single Molecule Studies ◽  
Ph Dependent

Most cellular ATP is made by rotary F1FO ATP synthases using proton translocation-generated clockwise torque on the FO c-ring rotor, while F1-ATP hydrolysis can force anticlockwise rotation and proton pumping. Although the interface of stator subunit-a containing the transmembrane half-channels and the c-ring is known from recent F1FO structures, the torque generating mechanism remains elusive. Here, single-molecule studies reveal pH-dependent 11° rotational sub-steps in the ATP synthase direction of the E. coli c10-ring of F1FO against the force of F1- ATPase-dependent rotation that result from H+ transfer events from FO subunit-a groups with a low pKa to one c-subunit of the c-ring, and from an adjacent c-subunit to stator groups with a high pKa. Mutations of subunit-a residues in the proton translocation channels alter these pKa values, and the ability of synthase substeps to occur. Alternating 11° and 25° sub-steps then result in sustained ATP synthase rotation of the c10-ring.

scholarly journals pH-dependent 11° F1FO ATP synthase sub-steps reveal insight into the FO torque generating mechanism

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Seiga Yanagisawa ◽  
Wayne D Frasch
Keyword(s):  
Atp Synthase ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Atp Synthesis ◽  
Proton Pumping ◽  
Subunit A ◽  
Pka Values ◽  
C Subunit ◽  
Ph Dependent

Most cellular ATP is made by rotary F1FO ATP synthases using proton translocation-generated clockwise torque on the FO c-ring rotor, while F1-ATP hydrolysis can force counterclockwise rotation and proton pumping. The FO torque-generating mechanism remains elusive even though the FO interface of stator subunit-a, which contains the transmembrane proton half-channels, and the c-ring is known from recent F1FO structures. Here, single-molecule F1FO rotation studies determined that the pKa values of the half-channels differ, show that mutations of residues in these channels change the pKa values of both half-channels, and reveal the ability of FO to undergo single c-subunit rotational stepping. These experiments provide evidence to support the hypothesis that proton translocation through FO operates via a Grotthuss mechanism involving a column of single water molecules in each half-channel linked by proton translocation-dependent c-ring rotation. We also observed pH-dependent 11° ATP synthase-direction sub-steps of the E. coli c10-ring of F1FO against the torque of F1-ATPase-dependent rotation that result from H+ transfer events from FO subunit-a groups with a low pKa to one c-subunit in the c-ring, and from an adjacent c-subunit to stator groups with a high pKa. These results support a mechanism in which alternating proton translocation-dependent 11° and 25° synthase-direction rotational sub-steps of the c10-ring occur to sustain F1FO ATP synthesis.

scholarly journals Intragenic and intergenic suppression of the Escherichia coli ATP synthase subunit a mutation of Gly-213 to Asn: functional interactions between residues in the proton transport site

2000 ◽  
Vol 347 (3) ◽  
pp. 797-805 ◽  
Author(s):  
Phillip H. KUO ◽  
Robert K. NAKAMOTO
Keyword(s):  
Transition State ◽  
Atp Synthase ◽  
Proton Transport ◽  
Atp Hydrolysis ◽  
Transmembrane Helix ◽  
Atp Synthesis ◽  
Hydrolytic Activity ◽  
Proton Pumping ◽  
Subunit A ◽  
Efficient Coupling

Subunit a of the ATP synthase Fo sector contains a transmembrane helix that interacts with subunit c and is critical for H+ transport activity. From a cysteine scan in the region around the essential subunit a residue, Arg-210, we found that the replacement of aGly-213 greatly attenuated ATP hydrolysis, ATP-dependent proton pumping and ∆μH+-dependent ATP synthesis. Various amino acid substitutions caused similar effects, suggesting that functional perturbations were caused by altering the environment or conformation of aArg-210. aG213N, which was particularly severe in effect, was suppressed by two second-site mutations, aL251V and cD61E. These mutations restored efficient coupling; the latter also increased ATP-dependent proton transport rates. These results were consistent with the proposed functional interaction between aArg-210 and cAsp-61, the likely carrier of the transported proton. From Arrhenius analysis of steady-state ATP hydrolytic activity, the transport mutants had large increases in the transition-state enthalpic and entropic parameters. Linear isokinetic relationships demonstrate that the transport mechanism is coupled to the rate-limiting catalytic transition-state step, which we have previously shown to involve the rotation of the γ subunit in multi-site, co-operative catalysis.

scholarly journals Structure of a bacterial ATP synthase

2018 ◽  
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.

scholarly journals What can be learned about the enzyme ATPase from single-molecule studies of its subunit F1?

2017 ◽  
Vol 50 ◽  
Author(s):  
Sándor Volkán-Kacso ◽  
Rudolph A. Marcus
Keyword(s):  
Atp Synthase ◽  
Single Molecule ◽  
Mechanical Theory ◽  
Different Types ◽  
Single Molecule Studies ◽  
Biological Motors

AbstractWe summarize the different types of single molecule experiments on the F1 component of FOF1-ATP Synthase and what has been learned from them. We also describe results from our recent studies on interpreting the experiments using a chemical-mechanical theory for these biological motors.

scholarly journals Structure and activation mechanism of the hexameric plasma membrane H+-ATPase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Peng Zhao ◽  
Chaoran Zhao ◽  
Dandan Chen ◽  
Caihong Yun ◽  
Huilin Li ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Conformational Changes ◽  
Liquid Crystalline ◽  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Proton Pumping ◽  
Antifungal Drugs ◽  
Activation Mechanism ◽  
Activation Process ◽  
Α Helix

AbstractThe S. cerevisiae plasma membrane H+-ATPase, Pma1, is a P3A-type ATPase and the primary protein component of the membrane compartment of Pma1 (MCP). Like other plasma membrane H+-ATPases, Pma1 assembles and functions as a hexamer, a property unique to this subfamily among the larger family of P-type ATPases. It has been unclear how Pma1 organizes the yeast membrane into MCP microdomains, or why it is that Pma1 needs to assemble into a hexamer to establish the membrane electrochemical proton gradient. Here we report a high-resolution cryo-EM study of native Pma1 hexamers embedded in endogenous lipids. Remarkably, we found that the Pma1 hexamer encircles a liquid-crystalline membrane domain composed of 57 ordered lipid molecules. The Pma1-encircled lipid patch structure likely serves as the building block of the MCP. At pH 7.4, the carboxyl-terminal regulatory α-helix binds to the phosphorylation domains of two neighboring Pma1 subunits, locking the hexamer in the autoinhibited state. The regulatory helix becomes disordered at lower pH, leading to activation of the Pma1 hexamer. The activation process is accompanied by a 6.7 Å downward shift and a 40° rotation of transmembrane helices 1 and 2 that line the proton translocation path. The conformational changes have enabled us to propose a detailed mechanism for ATP-hydrolysis-driven proton pumping across the plasma membrane. Our structures will facilitate the development of antifungal drugs that target this essential protein.

scholarly journals Structure of a bacterial ATP synthase

eLife ◽  
2019 ◽  
Vol 8 ◽  
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

ATP 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 the Bacillus PS3 ATP synthase in Eschericia 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 subunit ε shows 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.

scholarly journals Interaction of dibutyltin-3-hydroxyflavone bromide with the 16 kDa proteolipid indicates the disposition of proton translocation sites of the vacuolar ATPase

1996 ◽  
Vol 317 (2) ◽  
pp. 425-431 ◽  
Author(s):  
Glen HUGHES ◽  
Michael A. HARRISON ◽  
Yong-In KIM ◽  
David E. GRIFFITHS ◽  
Malcolm E. FINBOW ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Binding Sites ◽  
Fluorescence Enhancement ◽  
Atp Hydrolysis ◽  
Dissociation Constants ◽  
Proton Translocation ◽  
Vacuolar Atpase ◽  
Proton Pumping ◽  
Wild Type ◽  
Expression Studies

The organotin complex dibutyltin-3-hydroxyflavone bromide [Bu2Sn(of)Br] has been shown to bind to the 16 kDa proteolipid of Nephrops norvegicus, either in the form of the native protein or after heterologous expression in Saccharomyces and assembly into a hybrid vacuolar H+-ATPase. Titration of Bu2Sn(of)Br against the 16 kDa proteolipid results in a marked fluorescence enhancement, consistent with binding to a single affinity site on the protein. Vacuolar ATPase-dependent ATP hydrolysis was also inhibited by Bu2Sn(of)Br, with the inhibition constant correlating well with dissociation constants determined for binding of Bu2Sn(of)Br complex to the proteolipid. The fluorescence enhancement produced by interaction of probe with proteolipid can be back-titrated by dicyclohexylcarbodiimide (DCCD), which covalently modifies Glu140 on helix-4 of the polypeptide. Expression of a mutant proteolipid in which Glu140 was changed to a glycine resulted in assembly of a vacuolar ATPase which was inactive in proton pumping and which had reduced ATPase activity. Co-expression studies with this mutant and wild-type proteolipids suggest that proton pumping can only occur in a vacuolar ATPase containing exclusively wild-type proteolipid. The fluorescent enhancement or affinity of Bu2Sn(of)Br for the mutant proteolipid was not significantly altered, with the organotin complex having no effect on residual ATPase activity. Interaction of the probe with mutant proteolipid was unaffected by DCCD. These data suggest an overlap in the binding sites for organotin and DCCD, and have implications for the organization and structure of proton-translocating pathways in the vacuolar H+-ATPase.

scholarly journals Fast ATP-dependent subunit rotation in reconstituted FoF1-ATP synthase trapped in solution

2021 ◽  
Author(s):  
Thomas Heitkamp ◽  
Michael Börsch
Keyword(s):  
Atp Synthase ◽  
Single Molecule ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Atp Synthesis ◽  
Electrochemical Potential ◽  
Ensemble Averaging ◽  
Subunit Rotation ◽  
Atp Synthases

ABSTRACTFoF1-ATP synthases are the ubiquitous membrane enzymes which catalyze ATP synthesis or ATP hydrolysis in reverse, respectively. Enzyme kinetics are controlled by internal subunit rotation, by substrate and product concentrations, by mechanical inhibitory mechanisms, but also by the electrochemical potential of protons across the membrane. By utilizing an Anti- Brownian electrokinetic trap (ABEL trap), single-molecule Förster resonance energy transfer (smFRET)-based subunit rotation monitoring was prolonged from milliseconds to seconds. The extended observation times for single proteoliposomes in solution allowed to observe fluctuating rotation rates of individual enzymes and to map the broad distributions of ATP-dependent catalytic rates in FoF1-ATP synthase. The buildup of an electrochemical potential of protons was confirmed to limit the maximum rate of ATP hydrolysis. In the presence of ionophores and uncouplers the fastest subunit rotation speeds measured in single reconstituted FoF1-ATP synthases were 180 full rounds per second, i.e. much faster than measured by biochemical ensemble averaging, but not as fast as the maximum rotational speed reported previously for isolated single F1 fragments without coupling to the membrane-embedded Fo domain of the enzyme.

scholarly journals Molecular dynamics simulation of proton-transfer coupled rotations in ATP synthase FO motor

2019 ◽  
Author(s):  
Shintaroh Kubo ◽  
Toru Niina ◽  
Shoji Takada
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulation ◽  
Proton Transfer ◽  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Simulation Method ◽  
Proton Pumping ◽  
Dynamics Simulation ◽  
Transduction Efficiency

AbstractThe FO motor in FOF1 ATP synthase rotates its rotor driven by the proton motive force. While earlier studies elucidated basic mechanisms therein, recent advances in high-resolution cryo-electron microscopy enabled to investigate proton-transfer coupled FO rotary dynamics at structural details. Here, developing a hybrid Monte Carlo/molecular dynamics simulation method, we studied reversible dynamics of a yeast mitochondrial FO. We obtained the 36°-stepwise rotations of FO per one proton transfer in the ATP synthesis mode and the proton pumping in the ATP hydrolysis mode. In both modes, the most prominent path alternatively sampled states with two and three deprotonated glutamates in c-ring, by which the c-ring rotates one step. The free energy transduction efficiency in the model FO motor reaches ~ 90% in optimal conditions. Moreover, mutations in key glutamate and a highly conserved arginine increased proton leakage and markedly decreased the coupling, in harmony with previous experiments.

scholarly journals The Unique C-Terminal Extension of Mycobacterial F-ATP Synthase Subunit α Is the Major Contributor to Its Latent ATP Hydrolysis Activity

2020 ◽  
Vol 64 (12) ◽  
Author(s):  
Chui-Fann Wong ◽  
Gerhard Grüber
Keyword(s):  
Atpase Activity ◽  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Proton Motive Force ◽  
Terminal Deletion ◽  
Deletion Mutants ◽  
Major Contributor ◽  
Hydrolysis Activity ◽  
Binding Subunit

ABSTRACT Mycobacterial F1Fo-ATP synthases (α3:β3:γ:δ:ε:a:b:b′:c9) are incapable of ATP-driven proton translocation due to their latent ATPase activity. This prevents wasting of ATP and altering of the proton motive force, whose dissipation is lethal to mycobacteria. We demonstrate that the mycobacterial C-terminal extension of nucleotide-binding subunit α contributes mainly to the suppression of ATPase activity in the recombinant mycobacterial F1-ATPase. Using C-terminal deletion mutants, the regions responsible for the enzyme’s latency were mapped, providing a new compound epitope.

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