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 ATP synthase K+- and H+-flux drive ATP synthesis and enable mitochondrial K+-uniporter function

2018 ◽  
Author(s):  
Magdalena Juhaszova ◽  
Evgeny Kobrinsky ◽  
Dmitry B. Zorov ◽  
H. Bradley Nuss ◽  
Yael Yaniv ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Single Molecule ◽  
Energy Supply ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Atp Synthesis ◽  
Mechanical Efficiency ◽  
Major Fraction ◽  
Isolated Mitochondria ◽  
Related Proteins

SummaryATP synthase (F1Fo) synthesizes daily our body’s weight in ATP, whose production-rate can be transiently increased several-fold. Using purified mammalian F1Fo-reconstituted proteoliposomes and isolated mitochondria, we show that F1Fo utilizes both H+- and K+-transport (because of >106-fold K+ excess vs H+) to drive ATP synthesis with the H+:K+ permeability of ~106:1. F1Fo can be upregulated by endogenous survival-related proteins (Bcl-xL, Mcl-1) and synthetic molecules (diazoxide, pinacidil) to increase its chemo-mechanical efficiency via IF1. Increasing K+- and H+-driven ATP synthesis enables F1Fo to operate as a primary mitochondrial K+-uniporter regulating energy supply-demand matching, and as the recruitable mitochondrial KATP-channel that can limit ischemia-reperfusion injury. Isolated mitochondria in the presence of K+ can sustain ~3.5-fold higher ATP-synthesis-flux (vs K+ absence) driven by a 2.7:1 K+:H+ stoichiometry with unaltered OxPhos coupling. Excellent agreement between F1Fo single-molecule and intact-mitochondria experiments is consistent with K+-transport through ATP synthase driving a major fraction of ATP synthesis.

scholarly journals Re-Understanding the Mechanisms of Action of the Anti-Mycobacterial Drug Bedaquiline

Antibiotics ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 261 ◽  
Author(s):  
Jickky Palmae Sarathy ◽  
Gerhard Gruber ◽  
Thomas Dick
Keyword(s):  
Electron Transport ◽  
Atp Synthase ◽  
Atp Synthesis ◽  
Mechanisms Of Action ◽  
Direct Mechanism ◽  
Transport Chain ◽  
Ring Rotation ◽  
New Findings ◽  
Atp Generation ◽  
Resistance Mutants

Bedaquiline (BDQ) inhibits ATP generation in Mycobacterium tuberculosis by interfering with the F-ATP synthase activity. Two mechanisms of action of BDQ are broadly accepted. A direct mechanism involves BDQ binding to the enzyme’s c-ring to block its rotation, thus inhibiting ATP synthesis in the enzyme’s catalytic α3β3-headpiece. An indirect mechanism involves BDQ uncoupling electron transport in the electron transport chain from ATP synthesis at the F-ATP synthase. In a recently uncovered second direct mechanism, BDQ binds to the enzyme’s ε-subunit to disrupt its ability to link c-ring rotation to ATP synthesis at the α3β3-headpiece. However, this mechanism is controversial as the drug’s binding affinity for the isolated ε-subunit protein is moderate and spontaneous resistance mutants in the ε-subunit cannot be isolated. Recently, the new, structurally distinct BDQ analogue TBAJ-876 was utilized as a chemical probe to revisit BDQ’s mechanisms of action. In this review, we first summarize discoveries on BDQ’s mechanisms of action and then describe the new insights derived from the studies of TBAJ-876. The TBAJ-876 investigations confirm the c-ring as a target, while also supporting a functional role for targeting the ε-subunit. Surprisingly, the new findings suggest that the uncoupler mechanism does not play a key role in BDQ’s anti-mycobacterial activity.

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 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 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.

Stepwise rotation of the γ-subunit of EF o F 1 -ATP synthase during ATP synthesis: a single-molecule FRET approach

2003 ◽  
Author(s):  
Michael Borsch ◽  
Manuel Diez ◽  
Boris Zimmermann ◽  
Matthias Trost ◽  
Stefan Steigmiller ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Single Molecule ◽  
Atp Synthesis ◽  
Single Molecule Fret ◽  
Γ Subunit

scholarly journals Rates of various reactions catalyzed by ATP synthase as related to the mechanism of ATP synthesis.

1991 ◽  
Vol 266 (1) ◽  
pp. 123-129
Author(s):  
D A Berkich ◽  
G D Williams ◽  
P T Masiakos ◽  
M B Smith ◽  
P D Boyer ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Atp Synthesis

scholarly journals Carbon and Nitrogen Sources Have No Impact on the Organization and Composition of Ustilago maydis Respiratory Supercomplexes

2021 ◽  
Vol 7 (1) ◽  
pp. 42
Author(s):  
Deyamira Matuz-Mares ◽  
Oscar Flores-Herrera ◽  
Guadalupe Guerra-Sánchez ◽  
Lucero Romero-Aguilar ◽  
Héctor Vázquez-Meza ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Atp Synthesis ◽  
Nitrogen Sources ◽  
Growth Conditions ◽  
Energy Conditions ◽  
Respiratory Complexes ◽  
Respiratory Supercomplexes ◽  
Nadh Dehydrogenases ◽  
Reduced Nicotinamide Adenine Dinucleotide

Respiratory supercomplexes are found in mitochondria of eukaryotic cells and some bacteria. A hypothetical role of these supercomplexes is electron channeling, which in principle should increase the respiratory chain efficiency and ATP synthesis. In addition to the four classic respiratory complexes and the ATP synthase, U. maydis mitochondria contain three type II NADH dehydrogenases (NADH for reduced nicotinamide adenine dinucleotide) and the alternative oxidase. Changes in the composition of the respiratory supercomplexes due to energy requirements have been reported in certain organisms. In this study, we addressed the organization of the mitochondrial respiratory complexes in U. maydis under diverse energy conditions. Supercomplexes were obtained by solubilization of U. maydis mitochondria with digitonin and separated by blue native polyacrylamide gel electrophoresis (BN-PAGE). The molecular mass of supercomplexes and their probable stoichiometries were 1200 kDa (I1:IV1), 1400 kDa (I1:III2), 1600 kDa (I1:III2:IV1), and 1800 kDa (I1:III2:IV2). Concerning the ATP synthase, approximately half of the protein is present as a dimer and half as a monomer. The distribution of respiratory supercomplexes was the same in all growth conditions. We did not find evidence for the association of complex II and the alternative NADH dehydrogenases with other respiratory complexes.

scholarly journals Crystallographic structure of the turbine C-ring from spinach chloroplast F-ATP synthase

2014 ◽  
Vol 34 (2) ◽  
Author(s):  
Asha Manikkoth Balakrishna ◽  
Holger Seelert ◽  
Sven-Hendric Marx ◽  
Norbert A. Dencher ◽  
Gerhard Grüber
Keyword(s):  
Atp Synthase ◽  
Ring Width ◽  
Atp Synthesis ◽  
Crystallographic Structure ◽  
Unit Cell Parameters ◽  
Subunit C ◽  
Cell Parameters ◽  
Ring Diameter ◽  
C Subunit

In eukaryotic and prokaryotic cells, F-ATP synthases provide energy through the synthesis of ATP. The chloroplast F-ATP synthase (CF1FO-ATP synthase) of plants is integrated into the thylakoid membrane via its FO-domain subunits a, b, b’ and c. Subunit c with a stoichiometry of 14 and subunit a form the gate for H+-pumping, enabling the coupling of electrochemical energy with ATP synthesis in the F1 sector. Here we report the crystallization and structure determination of the c14-ring of subunit c of the CF1FO-ATP synthase from spinach chloroplasts. The crystals belonged to space group C2, with unit-cell parameters a=144.420, b=99.295, c=123.51 Å, and β=104.34° and diffracted to 4.5 Å resolution. Each c-ring contains 14 monomers in the asymmetric unit. The length of the c-ring is 60.32 Å, with an outer ring diameter 52.30 Å and an inner ring width of 40 Å.

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