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Rotary catalysis of FoF1-ATP synthase.

ABSTRACT: The synthesis of ATP, the key reaction of biological energy metabolism, is accomplished by the rotary motor protein; FoF1-ATP synthase (FoF1). In vivo, FoF1, located on the cell membrane, carries out ATP synthesis by using the proton motive force. This heterologous energy conversion is supposed to be mediated by the mechanical rotation of FoF1; however, it still remained unclear. Recently, we developed the novel experimental setup to reproduce the proton motive force in vitro and succeeded in directly observing the proton-driven rotation of FoF1. In this review, we describe the interesting working principles determined so far for FoF1 and then introduce results from our recent study.

SUBMITTER: Watanabe R

PROVIDER: S-EPMC4629669 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Rotary catalysis of FoF1-ATP synthase.

Watanabe Rikiya R

Biophysics (Nagoya-shi, Japan) 20130522

The synthesis of ATP, the key reaction of biological energy metabolism, is accomplished by the rotary motor protein; FoF1-ATP synthase (FoF1). In vivo, FoF1, located on the cell membrane, carries out ATP synthesis by using the proton motive force. This heterologous energy conversion is supposed to be mediated by the mechanical rotation of FoF1; however, it still remained unclear. Recently, we developed the novel experimental setup to reproduce the proton motive force in vitro and succeeded in di ...[more]

PMID: 27493540

Similar Datasets

ATP-driven stepwise rotation of FoF1-ATP synthase.

Project description:FoF1-ATP synthase (FoF1) is a motor enzyme that couples ATP synthesis/hydrolysis with a transmembrane proton translocation. F1, a water-soluble ATPase portion of FoF1, rotates by repeating ATP-waiting dwell, 80 degrees substep rotation, catalytic dwell, and 40 degrees -substep rotation. Compared with F1, rotation of FoF1 has yet been poorly understood, and, here, we analyzed ATP-driven rotations of FoF1. Rotation was probed with an 80-nm bead attached to the ring of c subunits in the immobilized FoF1 and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a Km of approximately 30 microM and a Vmax of approximately 350 revolutions per second (at 37 degrees C). At low ATP, ATP-waiting dwell was seen and the kon-ATP was estimated to be 3.6 x 10(7) M(-1) x s(-1). At high ATP, fast, poorly defined stepwise motions were observed that probably reflect the catalytic dwells. When a slowly hydrolyzable substrate, adenosine 5'-[gamma-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of approximately 80 degrees . The rotational behavior of FoF1 resembles that of F1. This finding indicates that "friction" in Fo motor is negligible during the ATP-driven rotation. Tributyltin chloride, a specific inhibitor of proton translocation, slowed the rotation rate by 96%. However, dwells at clearly defined angular positions were not observed under these conditions, indicating that inhibition by tributyltin chloride is complex.

| S-EPMC545493 | biostudies-literature

The regulatory subunit ? in Escherichia coli FOF1-ATP synthase.

Project description:F-type ATP synthases are extraordinary multisubunit proteins that operate as nanomotors. The Escherichia coli (E. coli) enzyme uses the proton motive force (pmf) across the bacterial plasma membrane to drive rotation of the central rotor subunits within a stator subunit complex. Through this mechanical rotation, the rotor coordinates three nucleotide binding sites that sequentially catalyze the synthesis of ATP. Moreover, the enzyme can hydrolyze ATP to turn the rotor in the opposite direction and generate pmf. The direction of net catalysis, i.e. synthesis or hydrolysis of ATP, depends on the cell's bioenergetic conditions. Different control mechanisms have been found for ATP synthases in mitochondria, chloroplasts and bacteria. This review discusses the auto-inhibitory behavior of subunit ? found in FOF1-ATP synthases of many bacteria. We focus on E. coli FOF1-ATP synthase, with insights into the regulatory mechanism of subunit ? arising from structural and biochemical studies complemented by single-molecule microscopy experiments.

| S-EPMC6114093 | biostudies-literature

Fo-driven Rotation in the ATP Synthase Direction against the Force of F1 ATPase in the FoF1 ATP Synthase.

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Nitration of specific tyrosines in FoF1 ATP synthase and activity loss in aging.

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An alternative role of FoF1-ATP synthase in Escherichia coli: synthesis of thiamine triphosphate.

Project description:In E. coli, thiamine triphosphate (ThTP), a putative signaling molecule, transiently accumulates in response to amino acid starvation. This accumulation requires the presence of an energy substrate yielding pyruvate. Here we show that in intact bacteria ThTP is synthesized from free thiamine diphosphate (ThDP) and P(i), the reaction being energized by the proton-motive force (?p) generated by the respiratory chain. ThTP production is suppressed in strains carrying mutations in F(1) or a deletion of the atp operon. Transformation with a plasmid encoding the whole atp operon fully restored ThTP production, highlighting the requirement for F(o)F(1)-ATP synthase in ThTP synthesis. Our results show that, under specific conditions of nutritional downshift, F(o)F(1)-ATP synthase catalyzes the synthesis of ThTP, rather than ATP, through a highly regulated process requiring pyruvate oxidation. Moreover, this chemiosmotic mechanism for ThTP production is conserved from E. coli to mammalian brain mitochondria.

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Visualizing Mitochondrial FoF1-ATP Synthase as the Target of the Immunomodulatory Drug Bz-423.

Project description:Targeting the mitochondrial enzyme FoF1-ATP synthase and modulating its catalytic activities with small molecules is a promising new approach for treatment of autoimmune diseases. The immunomodulatory compound Bz-423 is such a drug that binds to subunit OSCP of the mitochondrial FoF1-ATP synthase and induces apoptosis via increased reactive oxygen production in coupled, actively respiring mitochondria. Here, we review the experimental progress to reveal the binding of Bz-423 to the mitochondrial target and discuss how subunit rotation of FoF1-ATP synthase is affected by Bz-423. Briefly, we report how Förster resonance energy transfer can be employed to colocalize the enzyme and the fluorescently tagged Bz-423 within the mitochondria of living cells with nanometer resolution.

| S-EPMC6039829 | biostudies-literature

36 degrees step size of proton-driven c-ring rotation in FoF1-ATP synthase.

Project description:Synthesis of adenosine triphosphate ATP, the 'biological energy currency', is accomplished by F(o)F(1)-ATP synthase. In the plasma membrane of Escherichia coli, proton-driven rotation of a ring of 10 c subunits in the F(o) motor powers catalysis in the F(1) motor. Although F(1) uses 120 degrees stepping during ATP synthesis, models of F(o) predict either an incremental rotation of c subunits in 36 degrees steps or larger step sizes comprising several fast substeps. Using single-molecule fluorescence resonance energy transfer, we provide the first experimental determination of a 36 degrees sequential stepping mode of the c-ring during ATP synthesis.

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Double-lock ratchet mechanism revealing the role of alphaSER-344 in FoF1 ATP synthase.

Project description:In a majority of living organisms, FoF1 ATP synthase performs the fundamental process of ATP synthesis. Despite the simple net reaction formula, ADP+Pi?ATP+H2O, the detailed step-by-step mechanism of the reaction yet remains to be resolved owing to the complexity of this multisubunit enzyme. Based on quantum mechanical computations using recent high resolution X-ray structures, we propose that during ATP synthesis the enzyme first prepares the inorganic phosphate for the ?P-OADP bond-forming step via a double-proton transfer. At this step, the highly conserved ?S344 side chain plays a catalytic role. The reaction thereafter progresses through another transition state (TS) having a planar ion configuration to finally form ATP. These two TSs are concluded crucial for ATP synthesis. Using stepwise scans and several models of the nucleotide-bound active site, some of the most important conformational changes were traced toward direction of synthesis. Interestingly, as the active site geometry progresses toward the ATP-favoring tight binding site, at both of these TSs, a dramatic increase in barrier heights is observed for the reverse direction, i.e., hydrolysis of ATP. This change could indicate a "ratchet" mechanism for the enzyme to ensure efficacy of ATP synthesis by shifting residue conformation and thus locking access to the crucial TSs.

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Elasticity, friction, and pathway of γ-subunit rotation in FoF1-ATP synthase.

Project description:We combine molecular simulations and mechanical modeling to explore the mechanism of energy conversion in the coupled rotary motors of FoF1-ATP synthase. A torsional viscoelastic model with frictional dissipation quantitatively reproduces the dynamics and energetics seen in atomistic molecular dynamics simulations of torque-driven γ-subunit rotation in the F1-ATPase rotary motor. The torsional elastic coefficients determined from the simulations agree with results from independent single-molecule experiments probing different segments of the γ-subunit, which resolves a long-lasting controversy. At steady rotational speeds of ∼ 1 kHz corresponding to experimental turnover, the calculated frictional dissipation of less than k(B)T per rotation is consistent with the high thermodynamic efficiency of the fully reversible motor. Without load, the maximum rotational speed during transitions between dwells is reached at ∼ 1 MHz. Energetic constraints dictate a unique pathway for the coupled rotations of the Fo and F1 rotary motors in ATP synthase, and explain the need for the finer stepping of the F1 motor in the mammalian system, as seen in recent experiments. Compensating for incommensurate eightfold and threefold rotational symmetries in Fo and F1, respectively, a significant fraction of the external mechanical work is transiently stored as elastic energy in the γ-subunit. The general framework developed here should be applicable to other molecular machines.

| S-EPMC4553772 | biostudies-other

Time-delayed in vivo assembly of subunit a into preformed Escherichia coli FoF1 ATP synthase.

Project description:Escherichia coli F(O)F(1) ATP synthase, a rotary nanomachine, is composed of eight different subunits in a ?3?3???ab2c10 stoichiometry. Whereas F(O)F(1) has been studied in detail with regard to its structure and function, much less is known about how this multisubunit enzyme complex is assembled. Single-subunit atp deletion mutants are known to be arrested in assembly, thus leading to formation of partially assembled subcomplexes. To determine whether those subcomplexes are preserved in a stable standby mode, a time-delayed in vivo assembly system was developed. To establish this approach, we targeted the time-delayed assembly of membrane-integrated subunit a into preformed F(O)F(1) lacking subunit a (F(O)F(1)-a) which is known to form stable subcomplexes in vitro. Two expression systems (araBADp and T7p-laco) were adjusted to provide compatible, mutually independent, and sufficiently stringent induction and repression regimens. In detail, all structural atp genes except atpB (encoding subunit a) were expressed under the control of araBADp and induced by arabinose. Following synthesis of F(O)F(1)-a during growth, expression was repressed by glucose/d-fucose, and degradation of atp mRNA controlled by real-time reverse transcription-PCR. A time-delayed expression of atpB under T7p-laco control was subsequently induced in trans by addition of isopropyl-?-d-thiogalactopyranoside. Formation of fully assembled, and functional, F(O)F(1) complexes was verified. This demonstrates that all subunits of F(O)F(1)-a remain in a stable preformed state capable to integrate subunit a as the last subunit. The results reveal that the approach presented here can be applied as a general method to study the assembly of heteromultimeric protein complexes in vivo.

| S-EPMC3754730 | biostudies-literature

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