ATP synthesis by F-type ATP synthase is obligatorily dependent on the transmembrane voltage.

ATP synthase is the universal enzyme that manufactures cellular ATP using the energy stored in a transmembrane ion gradient. This energy gradient has two components: the concentration difference (DeltapH or DeltapNa(+)) and the electrical potential difference DeltaPsi, which are thermodynamically equivalent. However, they are not kinetically equivalent, as the mitochondrial and bacterial ATP synthases require a transmembrane potential, DeltaPsi, but the chloroplast enzyme has appeared to operate on DeltapH alone. Here we show that, contrary to the accepted wisdom, the 'acid bath' procedure used to study the chloroplast enzyme develops not only a DeltapH but also a membrane potential, and that this potential is essential for ATP synthesis. Thus, for the chloroplast and other ATP synthases, the membrane potential is the fundamental driving force for their normal operation. We discuss the biochemical reasons for this phenomenon and a model that is consistent with these new experimental facts.     

Critical evaluation of the one- versus the two-channel model for the operation of the ATP synthase's F(o) motor

The mechanism of converting an electrochemical gradient of protons or Na(+) ions across the membrane into rotational torque by the F(o) motor of the ATP synthase has been described by a two-channel model or by a one-channel model. Experimental evidence obtained with the F(o) motor from the Propionigenium modestum ATP synthase is described which is in accordance with the one-channel model, but not with the two-channel model. This evidence includes the ATP-dependent occlusion of one (22)Na(+) per ATP synthase with a mutated Na(+)-impermeable a subunit or the Na(+)(in)/(22)Na(+)(out) exchange which is not affected by modifying part of the c subunit sites with dicyclohexylcarbodiimide.     

Biological nano motor, ATP synthase F(o)F(1): from catalysis to gammaepsilonc(10-12) subunit assembly rotation

Proton translocating ATPase (ATP synthase), a chemiosmotic enzyme, synthesizes ATP from ADP and phosphate coupling with the electrochemical ion gradient across the membrane. This enzyme has been studied extensively by combined genetic, biochemical and biophysical approaches. Such studies revealed a unique mechanism which transforms an electrochemical ion gradient into chemical energy through the rotation of a subunit assembly. Thus, this enzyme can be defined as a nano motor capable of coupling a chemical reaction and ion translocation, or more simply, as a protein complex carrying out rotational catalysis. In this article, we briefly discuss our recent work, emphasizing the rotation of subunit assembly (gammaepsilonc(10-12)) which is formed from peripheral and intrinsic membrane subunits.     

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

Living organisms rely on the F_oF₁ ATP synthase to maintain the non-equilibrium chemical gradient of ATP to ADP and phosphate that provides the primary energy source for cellular processes. How the F_o motor uses a transmembrane electrochemical ion gradient to create clockwise torque that overcomes F₁ ATPase-driven counterclockwise torque at high ATP is a major unresolved question. Using single F_oF₁ molecules embedded in lipid bilayer nanodiscs, we now report the observation of F_o-dependent rotation of the c10 ring in the ATP synthase (clockwise) direction against the counterclockwise force of ATPase-driven rotation that occurs upon formation of a leash with F_o stator subunit a. Mutational studies indicate that the leash is important for ATP synthase activity and support a mechanism in which residues aGlu-196 and cArg-50 participate in the cytoplasmic proton half-channel to promote leash formation.     

The Na(+)-translocating F(1)F(0) ATP synthase of Propionigenium modestum: mechanochemical insights into the F(0) motor that drives ATP synthesis

The ATP synthase of Propionigenium modestum encloses a rotary motor involved in the production of ATP from ADP and inorganic phosphate utilizing the free energy of an electrochemical Na(+) ion gradient. This enzyme clearly belongs to the family of F(1)F(0) ATP synthases and uses exclusively Na(+) ions as the physiological coupling ion. The motor domain, F(0), comprises subunit a and the b subunit dimer which are part of the stator and the subunit c oligomer acting as part of the rotor. During ATP synthesis, Na(+) translocation through F(0) proceeds from the periplasm via the stator channel (subunit a) onto a Na(+) binding site of the rotor (subunit c). Upon rotation of the subunit c oligomer versus subunit a, the occupied rotor site leaves the interface with the stator and the Na(+) ion can freely dissociate into the cytoplasm. Recent experiments demonstrate that the membrane potential is crucial for ATP synthesis under physiological conditions. These findings support the view that voltage generates torque in F(0), which drives the rotation of the gamma subunit thus liberating tightly bound ATP from the catalytic sites in F(1). We suggest a mechanochemical model for the transduction of transmembrane Na(+)-motive force into rotary torque by the F(0) motor that can account quantitatively for the experimental data.     

Delta mu Na+ drives the synthesis of ATP via an delta mu Na(+)-translocating F1F0-ATP synthase in membrane vesicles of the archaeon Methanosarcina mazei Gö1.

Methanosarcina mazei Gö1 couples the methyl transfer from methyl-tetrahydromethanopterin to 2-mercaptoethanesulfonate (coenzyme M) with the generation of an electrochemical sodium ion gradient (delta mu Na+) and the reduction of the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreoninephosphate with the generation of an electrochemical proton gradient (delta muH+). Experiments with washed inverted vesicles were performed to investigate whether both ion gradients are used directly for the synthesis of ATP. delta mu Na+ and delta mu H+ were both able to drive the synthesis of ATP in the vesicular system. ATP synthesis driven by heterodisulfide reduction (delta mu H+) or an artificial delta pH was inhibited by the protonophore SF6847 but not by the sodium ionophore ETH157, whereas ETH157 but not SF6847 inhibited ATP synthesis driven by a chemical sodium ion gradient (delta pNa) as well as the methyl transfer reaction (delta mu Na+). Inhibition of the Na+/H+ antiporter led to a stimulation of ATP synthesis driven by the methyl transfer reaction (delta mu Na+), as well as by delta pNa. These experiments indicate that delta mu Na+ and delta mu H+ drive the synthesis of ATP via an Na(+)- and an H(+)-translocating ATP synthase, respectively. Inhibitor studies were performed to elucidate the nature of the ATP synthase(s) involved. delta pH-driven ATP synthesis was specifically inhibited by bafilomycin A1, whereas delta pNa-driven ATP synthesis was exclusively inhibited by 7-chloro-4-nitro-2-oxa-1,3-diazole, azide, and venturicidin. These results are evidence for the presence of an F(1)F(0)-ATP synthase in addition to the A(1)A(0)-ATP synthase in membranes of M. Mazei Gö1 and suggest that the F(1)F(0)-type enzyme is an Na+-translocating ATP synthase, whereas the A(1)A(0)-ATP synthase uses H+ as the coupling ion.     

Two distinct proton binding sites in the ATP synthase family

The F1F0 ATP synthase utilizes energy stored in an electrochemical gradient of protons (or Na+ ions) across the membrane to synthesize ATP from ADP and phosphate. Current models predict that the protonation/deprotonation of specific acidic c ring residues is at the core of the proton translocation mechanism by this enzyme. To probe the mode of proton binding, we measured the covalent modification of the acidic c ring residues with the inhibitor dicyclohexylcarbodiimide (DCCD) over the pH range from 5 to 11. With the H+-translocating ATP synthase from the archaeum Halobacterium salinarium or the Na+-translocating ATP synthase from Ilyobacter tartaricus, the pH profile of DCCD labeling followed a titration curve with a pKa around neutral, reflecting protonation of the acidic c ring residues. However, with the ATP synthases from Escherichia coli, mitochondria, or chloroplasts, a clearly different, bell-shaped pH profile for DCCD labeling was observed which is not compatible with carboxylate protonation but might be explained by the coordination of a hydronium ion as proposed earlier [Boyer, P. D. (1988) Trends Biochem. Sci. 13, 5-7]. Upon site-directed mutagenesis of single binding site residues of the structurally resolved c ring, the sigmoidal pH profile for DCCD labeling could be converted to a more bell-shaped one, demonstrating that the different ion binding modes are based on subtle changes in the amino acid sequence of the protein. The concept of two different binding sites in the ATP synthase family is supported by the ATP hydrolysis pH profiles of the investigated enzymes.     

ATP合酶的结构与催化机理

ATP合酶 (F1Fo 复合物) 是生物体内进行氧化磷酸化和光合磷酸化的关键酶.随着核磁共振、X射线晶体衍射、遗传学、化学交联等技术在ATP合酶研究中的广泛应用,ATP合酶的整体结构及其各组成亚基结构的研究都有很大的进展.其中细菌ATP合酶结构的研究更为深入.目前对质子通过Fo的转运方式提出两种模型:单通道和双半通道模型.对扭力矩的形成以及旋转催化也有了进一步的认识.Boyer提出的结合改变机理推动了ATP合酶催化机制的研究,现在主要有两点催化机制和三点催化机制.ATP合酶的催化反应受酶的构象变化和外在条件的调节.     

Kinetic model of ATP synthase: pH dependence of the rate of ATP synthesis

Recently, a novel molecular mechanism of torque generation in the F(0) portion of ATP synthase was proposed [Rohatgi, Saha and Nath (1998) Curr. Sci. 75, 716-718]. In this mechanism, rotation of the c-subunit was conceived to take place in 12 discrete steps of 30 degrees each due to the binding and unbinding of protons to/from the leading and trailing Asp-61 residues of the c-subunit, respectively. Based on this molecular mechanism, a kinetic scheme has been developed in this work. The scheme considers proton transport driven by a concentration gradient of protons across the proton half-channels, and the rotation of the c-subunit by changes in the electrical potential only. This kinetic scheme has been analyzed mathematically and an expression has been obtained to explain the pH dependence of the rate of ATP synthesis by ATP synthase under steady state operating conditions. For a single set of three enzymological kinetic parameters, this expression predicts the rates of ATP synthesis which agree well with the experimental data over a wide range of pH(in) and pH(out). A logical consequence of our analysis is that DeltapH and Deltapsi are kinetically inequivalent driving forces for ATP synthesis.     

Purification and biochemical characterization of the F1Fo-ATP synthase from thermoalkaliphilic Bacillus sp. strain TA2.A1

We describe here purification and biochemical characterization of the F(1)F(o)-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F(1)F(o)-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F(1) subunits alpha, beta, gamma, delta, and epsilon and F(o) subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F(1)F(o) holoenzyme or the isolated F(1) moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F(1). After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Deltapsi). A transmembrane proton gradient or sodium ion gradient in the absence of Deltapsi was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na(+)/H(+) antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na(+) ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N'-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.     

Mitochondrial ATP synthase c-subunit leak channel triggers cell death upon loss of its F1 subcomplex

Mitochondrial ATP synthase is vital not only for cellular energy production but also for energy dissipation and cell death. ATP synthase c-ring was suggested to house the leak channel of mitochondrial permeability transition (mPT), which activates during excitotoxic ischemic insult. In this present study, we purified human c-ring from both eukaryotic and prokaryotic hosts to biophysically characterize its channel activity. We show that purified c-ring forms a large multi-conductance, voltage-gated ion channel that is inhibited by the addition of ATP synthase F₁ subcomplex. In contrast, dissociation of F₁ from F_O occurs during excitotoxic neuronal death suggesting that the F₁ constitutes the gate of the channel. mPT is known to dissipate the osmotic gradient across the inner membrane during cell death. We show that ATP synthase c-subunit knock down (KD) prevents the osmotic change in response to high calcium and eliminates large conductance, Ca²⁺ and CsA sensitive channel activity of mPT. These findings elucidate the gating mechanism of the ATP synthase c-subunit leak channel (ACLC) and suggest how ACLC opening is regulated by cell stress in a CypD-dependent manner.Subject terms: Cell biology, Neuroscience     

Reverse engineering a protein: the mechanochemistry of ATP synthase

ATP synthase comprises two rotary motors in one. The F(1) motor can generate a mechanical torque using the hydrolysis energy of ATP. The F(o) motor generates a rotary torque in the opposite direction, but it employs a transmembrane proton motive force. Each motor can be reversed: The F(o) motor can drive the F(1) motor in reverse to synthesize ATP, and the F(1) motor can drive the F(o) motor in reverse to pump protons. Thus ATP synthase exhibits two of the major energy transduction pathways employed by the cell to convert chemical energy into mechanical force. Here we show how a physical analysis of the F(1) and F(o) motors can provide a unified view of the mechanochemical principles underlying these energy transducers.     

Mechanism of the F(1)F(0)-type ATP synthase, a biological rotary motor

The F(1)F(0)-type ATP synthase is a key enzyme in cellular energy interconversion. During ATP synthesis, this large protein complex uses a proton gradient and the associated membrane potential to synthesize ATP. It can also reverse and hydrolyze ATP to generate a proton gradient. The structure of this enzyme in different functional forms is now being rapidly elucidated. The emerging consensus is that the enzyme is constructed as two rotary motors, one in the F(1) part that links catalytic site events with movements of an internal rotor, and the other in the F(0) part, linking proton translocation to movements of this F(0) rotor. Although both motors can work separately, they must be connected together to interconvert energy. Evidence for the function of the rotary motor, from structural, genetic and biophysical studies, is reviewed here, and some uncertainties and remaining mysteries of the enzyme mechanism are also discussed.     

The rotor in the membrane of the ATP synthase and relatives

In recent years, structural information on the F(1) sector of the ATP synthase has provided an insight into the molecular mechanism of ATP catalysis. The structure strongly supports the proposal that the ATP synthase works as a rotary molecular motor. Insights into the membrane domain have just started to emerge but more detailed structural information is needed if the molecular mechanism of proton translocation coupled to ATP synthesis is to be understood. This review will focus mainly on the ion translocating rotor in the membrane domain of the F-type ATPase, and the related vacuolar and archaeal relatives.     

Voltage-generated torque drives the motor of the ATP synthase.

The mechanism by which ion-flux through the membrane-bound motor module (F0) induces rotational torque, driving the rotation of the gamma subunit, was probed with a Na+-translocating hybrid ATP synthase. The ATP-dependent occlusion of 1 (22)Na+ per ATP synthase persisted after modification of the c subunit ring with dicyclohexylcarbodiimide (DCCD), when 22Na+ was added first and ATP second, but not if the order of addition was reversed. These results support the model of ATP-driven rotation of the c subunit oligomer (rotor) versus subunit a (stator) that stops when either a 22Na+-loaded or a DCCD-modified rotor subunit reaches the Na+-impermeable stator. The ATP synthase with a Na+-permeable stator catalyzed 22Na+out/Na+in-exchange after reconstitution into proteoliposomes, which was not significantly affected by DCCD modification of the c subunit oligomer, but was abolished by the additional presence of ATP or by a membrane potential (DeltaPsi) of 90 mV. We propose that in the idling mode of the motor, Na+ ions are shuttled across the membrane by limited back and forth movements of the rotor against the stator. This motional flexibility is arrested if either ATP or DeltaPsi induces the switch from idling into a directed rotation. The Propionigenium modestum ATP synthase catalyzed ATP formation with DeltaPsi of 60-125 mV but not with DeltapNa+ of 195 mV. These results demonstrate that electric forces are essential for ATP synthesis and lead to a new concept of rotary-torque generation in the ATP synthase motor.     

An intermediate step in the evolution of ATPases--the F1F0-ATPase from Acetobacterium woodii contains F-type and V-type rotor subunits and is capable of ATP synthesis

Previous preparations of the Na(+) F(1)F(0)-ATP synthase solubilized by Triton X-100 lacked some of the membrane-embedded motor subunits [Reidlinger J & Müller V (1994) Eur J Biochem233, 275-283]. To improve the subunit recovery, we revised our purification protocol. The ATP synthase was solubilized with dodecylmaltoside and further purified to apparent homogeneity by chromatographic techniques. The preparation contained, along with the F(1) subunits, the entire membrane-embedded motor with the stator subunits a and b, and the heterooligomeric c ring, which contained the V(1)V(0)-like subunit c(1) and the F(1)F(0)-like subunits c(2) and c(3). After incorporation into liposomes, ATP synthesis could be driven by an electrochemical sodium ion potential or a potassium ion diffusion potential, but not by a sodium ion potential. This is the first demonstration that an ATPase with a V(0)-F(0) hybrid motor is capable of ATP synthesis.     

Control of mitochondrial ATP synthase in rat cardiomyocytes: effects of thyroid hormone.

Activities of the mitochondrial ATP synthase and the electron transfer chain were investigated in cultured cardiomyocytes prepared from untreated and thyroxine-treated rats. Quiescent cells from the thyroxine-treated animals showed a 33% increase in mitochondrial ATP synthase capacity, but no change in respiratory chain capacity, relative to those from control animals. This increase was attributable largely to (a) a 25% increase in F1 content in these mitochondria, and partly to (b) a 10% stimulation in ATPase activity due to raised intramitochondrial Ca2+. Both types of cell showed a normal ATP content of 38-40 nmol/mg cell protein. In control cells, the mitochondrial ATP synthase responded to increased energy demand (by electrical stimulation and/or by positive inotropic agents) with an increase in its capacity of up to 2-fold. This response was absent in cells from thyroxine-treated animals. In addition, cellular ATP levels fell significantly after 2 min electrical stimulation of cells from thyroxine-treated animals, while those of control cells were constant. It was concluded that regulation of the mitochondrial ATP synthase was defective in heart cells from thyroxine treated rats, leading to an energy deficit when energy demand on the cells was increased. Animals treated with thyroxine, but allowed to recover for 17 days after treatment, showed responses indistinguishable from the control cells. Thus, the effects of thyroxine on mitochondrial activities were reversible.     

A Maxwell's Demon type of membrane transport: possibility for active transport by ABC-type transporters?

ATP-binding-cassette (or ABC)-type transporters constitute one of the largest family of membrane transporters in nature. Many of its members move substrates "actively", i.e. in an ATP-dependent manner against an electrochemical gradient. No consensus is available about the mechanism. Therefore, a novel class of transport mechanisms is proposed based on Maxwell's demon idea. This transport mechanism consists of a gated pore that selectively opens for substrates from one, but not the other side. Thermoenergy (Brownian motion) would suffice for substrate translocation across the membrane; energy for synchronizing gate opening with substrate arrival would come from ATP hydrolysis. Simulations demonstrate that such a mechanism would be thermodynamically and kinetically feasible. It exhibits "active", unidirectional transport, saturation, and other typical features of protein-catalysed reactions. It also shows pore behavior with charged substrates moving under the influence of electrical potentials. Its efficiency depends on a diffusion time constant of the substrate in solution that is slower than the transit time through the membrane, a situation that can realistically be achieved at millimolar or lower substrate concentrations. Features of the novel mechanism that differ significantly from P- or F-type ATPases are: (1) transport cannot be run in "reverse" to synthesize ATP even if sufficient energy is available in the gradient of the transported solute and (2) unidirectional and net substrate fluxes through the transporter diverge with increasing substrate concentration.     

Structure of dimeric ATP synthase from mitochondria: an angular association of monomers induces the strong curvature of the inner membrane

Respiration in all cells depends upon synthesis of ATP by the ATP synthase complex, a rotary motor enzyme. The structure of the catalytic moiety of ATP synthase, the so-called F(1) headpiece, is well established. F(1) is connected to the membrane-bound and ion translocating F(0) subcomplex by a central stalk. A peripheral stalk, or stator, prevents futile rotation of the headpiece during catalysis. Although the enzyme functions as a monomer, several lines of evidence have recently suggested that monomeric ATP synthase complexes might interact to form a dimeric supercomplex in mitochondria. However, due to its fragility, the structure of ATP synthase dimers has so far not been precisely defined for any organism. Here we report the purification of a stable dimeric ATP synthase supercomplex, using mitochondria of the alga Polytomella. Structural analysis by electron microscopy and single particle analysis revealed that dimer formation is based on specific interaction of the F(0) parts, not the F(1) headpieces which are not at all in close proximity. Remarkably, the angle between the two F(0) part is about 70 degrees, which induces a strong local bending of the membrane. Hence, the function of ATP synthase dimerisation is to control the unique architecture of the mitochondrial inner membrane.     

The molecular mechanism of ATP synthesis by F1F0-ATP synthase

ATP synthesis by oxidative phosphorylation and photophosphorylation, catalyzed by F1F0-ATP synthase, is the fundamental means of cell energy production. Earlier mutagenesis studies had gone some way to describing the mechanism. More recently, several X-ray structures at atomic resolution have pictured the catalytic sites, and real-time video recordings of subunit rotation have left no doubt of the nature of energy coupling between the transmembrane proton gradient and the catalytic sites in this extraordinary molecular motor. Nonetheless, the molecular events that are required to accomplish the chemical synthesis of ATP remain undefined. In this review we summarize current state of knowledge and present a hypothesis for the molecular mechanism of ATP synthesis.