Osmomechanics of the Propionigenium modestum Fo Motor (original) (raw)

2000, Journal of Bioenergetics and Biomembranes

https://doi.org/10.1023/A:1005608823087

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Abstract

In Propionigenium modestum, ATP is manufactured from ADP and phosphate by the enzyme ATP synthase using the free energy of an electrochemical gradient of Na+ ions. The P. modestum ATP synthase is a clear member of the family of F-type ATP synthases and the only major distinction is an extension of the coupling ion specificity to H+, Li+, or Na+,

Voltage-generated torque drives the motor of the ATP synthase

The EMBO Journal, 1998

The mechanism by which ion-flux through the membrane-bound motor module (F 0) induces rotational torque, driving the rotation of the γ 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 22 Na ⍣ 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 22 Na ⍣-loaded or a DCCD-modified rotor subunit reaches the Na ⍣-impermeable stator. The ATP synthase with a Na ⍣-permeable stator catalyzed 22 Na ⍣ 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 (∆Ψ) 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 ∆Ψ induces the switch from idling into a directed rotation. The Propionigenium modestum ATP synthase catalyzed ATP formation with ∆Ψ of 60-125 mV but not with ∆pNa ⍣ 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.

Molecular Mechanism of the ATP Synthase's Fo Motor Probed by Mutational Analyses of Subunit a

Journal of Molecular Biology, 2002

The most prominent residue of subunit a of the F 1 F o ATP synthase is a universally conserved arginine (aR227 in Propionigenium modestum ), which was reported to permit no substitution with retention of ATP synthesis or H þ -coupled ATP hydrolysis activity. We show here that ATP synthases with R227K or R227H mutations in the P. modestum a subunit catalyse ATP-driven Na þ transport above or below pH 8.0, respectively. Reconstituted F o with either mutation catalysed 22 Na þ out /Na þ in exchange with similar pH profiles as found in ATP-driven Na þ transport. ATP synthase with an aR227A substitution catalysed Na þ -dependent ATP hydrolysis, which was completely inhibited by dicyclohexylcarbodiimide, but not coupled to Na þ transport. This suggests that in the mutant the dissociation of Na þ becomes more difficult and that the alkali ions remain therefore permanently bound to the c subunit sites. The reconstituted mutant enzyme was also able to synthesise ATP in the presence of a membrane potential, which stopped at elevated external Na þ concentrations. These observations reinforce the importance of aR227 to facilitate the dissociation of Na þ from approaching rotor sites. This task of aR227 was corroborated by other results with the aR227A mutant: (i) after reconstitution into liposomes, F o with the aR227A mutation did not catalyse 22 Na þ out / Na þ in exchange at high internal sodium concentrations, and (ii) at a constant DpNa þ , 22 Na þ uptake was inhibited at elevated internal Na þ concentrations. Hence, in mutant aR227A, sodium ions can only dissociate from their rotor sites into a reservoir of low sodium ion concentration, whereas in the wild-type the positively charged aR227 allows the dissociation of Na þ even into compartments of high Na þ concentration.

ATP Synthase Motor Components: Proposal and Animation of Two Dynamic Models for Stator Function

Biochemical and Biophysical Research Communications, 2001

Recent research indicates that ATP synthases (F 0 F 1 ) contain two distinct nanomotors, one an electrochemically driven proton motor contained within F 0 that drives an ATP hydrolysis-driven motor (F 1 ) in reverse during ATP synthesis. This is depicted in recent models as involving a series of events in which each of the three ␣␤ pairs comprising F 1 is induced via a centrally rotating subunit (␥) to undergo the sequential binding changes necessary to synthesize ATP (binding change mechanism). Stabilization of this rotary process (i.e., to minimize "wobble" of F 1 ) is provided in current models by a peripheral stalk or "stator" that has recently been shown to extend from near the bottom of the ATP synthase molecule to the very top of F 1 . Although quite elegant, these models envision the stator as fixed during ATP synthesis, i.e., bound to only a single ␣␤ pair. This is despite the fact that the binding change mechanism views each ␣␤ pair as going through the same sequential order of conformational changes which demonstrate a chemical equivalency among them. For this reason, we propose here two different dynamic models for stator function during ATP synthesis. Both models have been designed to maintain chemical equivalency among the three ␣␤ pairs during ATP synthesis and both have been animated.

Energy transduction in the F1 motor of ATP synthase

ATP synthase is the universal enzyme that manufactures ATP from ADP and phosphate by using the energy derived from a transmembrane protonmotive gradient. It can also reverse itself and hydrolyse ATP to pump protons against an electrochemical gradient. ATP synthase carries out both its synthetic and hydrolytic cycles by a rotary mechanism 1±4 . This has been con®rmed in the direction of hydrolysis 5,6 after isolation of the soluble F 1 portion of the protein and visualization of the actual rotation of the central`shaft' of the enzyme with respect to the rest of the molecule, making ATP synthase the world's smallest rotary engine. Here we present a model for this engine that accounts for its mechanochemical behaviour in both the hydrolysing and synthesizing directions. We conclude that the F 1 motor achieves its high mechanical torque and almost 100% ef®ciency because it converts the free energy of ATP binding into elastic strain, which is then released by a coordinated kinetic and tightly coupled conformational mechanism to create a rotary torque.

Subunit c from the Sodium‐Ion‐Translocating F1F0‐ATPase of Propionigenium Modestum

European Journal of Biochemistry, 1997

Escherichia coli strain PEF42 produces a sodium‐ion‐dependent hybrid F1F0‐ATPase consisting of the Propionigenium modestum subunits a, b, c and δ, of a hybrid a subunit and of the E. coli subunits β, γ and E. The gene encoding subunit c of the P. modestum F1F0‐ATPase was cloned into the pT7‐7 expression vector to yield plasmid pT7c. E. coli PEF42 was transformed with plasmid pT7c together with plasmid pGP1–2, which harbours the gene for the T7 RNA polymerase. The production of the P. modestum subunit c was induced by a temperature shift from 30°C to 42°C for 30 min and led to an increased concentration of this protein in the membrane of the host strain. The c subunit produced in E. coli moved as a monomer in dodecylsulfate electrophoresis. The protein was extracted from the cells with chlorofodmethanol, purified and incorporated into sodium dodecylsulfate micelles. Circular dichroism of subunit c in sodium dodecylsulfate showed a temperature‐stable spectrum (between 20–60°C) with a ...

ATP synthase: two motors, two fuels

Structure, 1999

ATPase is the universal protein responsible for ATP synthesis. The enzyme comprises two reversible rotary motors: F o is either an ion 'turbine' or an ion pump, and F 1 is either a hydrolysis motor or an ATP synthesizer. Recent biophysical and biochemical studies have helped to elucidate the operating principles for both motors.

Electrical Power Fuels Rotary ATP Synthase

Structure, 2003

Institut fu ¨r Mikrobiologie der Eidgeno ¨ssischen Technischen Hochschule (Weber and Senior, 2003; Menz et al., 2001). Conversely, ATP hydrolysis by the F 1 motor causes reverse rotation ETH Zentrum CH-8092 Zu ¨rich of the shaft, which converts the F 0 motor into an ion pump. Under normal circumstances, the F 0 motor gener-Switzerland ates the larger torque and drives the F 1 motor in ATP synthesis direction. However, in anaerobically growing bacteria, when the respiratory enzymes are not active, ATP synthesis by F-type ATP synthases consumes the F 1 motor hydrolyzes ATP to use the F 0 motor as the energy stored in a transmembrane electrochemical generator of the indispensable membrane potential. gradient of protons or sodium ions. The electric com-The rotational model has gained impressive support ponent of the ion motive force is crucial for ATP synfrom the crystal structure of F 1 , which shows a marked thesis. Here, we incorporate recent results on strucasymmetry in the conformations and nucleotide occuture and function of the F 0 domain and present a pancy of the catalytic ␤ subunits (Abrahams et al., 1994). mechanism for torque generation with the fundamen-These structural features suggest that the different contal nature of the membrane potential as driving force formations interconvert by rotation of the central ␥ subin the core. unit relative to the (␣␤) 3 subcomplex. Once this structure was available, it guided experimental approaches to establish the rotational catalysis by a variety of biochemi-Energy conversions are central to all life forms. The cal and spectroscopic techniques. Most convincingly, degradation of nutrients in animals or bacteria or photothe rotation of a micrometer-sized fluorescent actin filasynthesis in plants culminates in the production of ATP, ment attached to the central shaft ␥ subunit has been the universal energy currency of living cells. The capacdirectly visualized by video microscopy of single F 1 molity of this process is impressive: the daily turnover of a ecules (Noji et al., 1997). human has been estimated to be 40 kg of ATP on aver-Construction of the F 0 Motor age. The central metabolic role of ATP has stimulated Structurally, the least well-defined part of the ATP synmuch interest in how it is formed using the energy of thase is F 0 . So far, no high-resolution structures of this oxidations or light. Research in this area has led to domain are known, which would undoubtedly be needed impressive progress, including some of the most specto explain the energy transduction mechanism in molectacular discoveries in the history of biochemistry. One ular terms. However, a wealth of biochemical knowledge of the highlights was Peter Mitchell's unprecedented about the driving forces used for rotation, the ion path recognition that energy derived from light or oxidation across the membrane, and the function of key amino could create an electrochemical proton gradient across acid residues is available to draw a good picture of the the chloroplast or mitochondrial membrane in which the F 0 motor. Based on moderate-resolution structural data, enzymes are embedded. The protonmotive force is then the overall architecture of F 0 consists of an oligomeric used to drive ATP synthesis from ADP and phosphate. ring of c subunits that is flanked laterally by a single This discovery shifted the focus to the question of how a subunit (Mellwig and Bo ¨ttcher, 2003). Subunit a is the movement of protons across the coupling memconnected by the peripheral stalk b 2 subunits to F 1 . The brane induced the formation of ATP and thus into the c ring together with the ␥ and ⑀ stalk comprises the core of the mechanism of the ATP-synthesizing enzyme. rotor. As ions traverse the membrane through the a-c The enzyme responsible for ATP synthesis is the F 1 F 0 interface, the rotor turns against the residual parts of ATP synthase, which is ubiquitous from bacteria to the assembly that is termed stator by convention. Most plants and animals. Our current knowledge of structure structural studies of F 0 were performed with subunit c. and function of an ATP synthase is shown in cartoon Each c subunit is folded as two transmembrane ␣ helices form in Figure 1. The multisubunit enzyme consists of connected by a loop (Girvin et al., 1998). In the oligomeric two domains, F 0 and F 1 , which are connected by a central assembly, the N-terminal helices pack very tightly into and a peripheral stalk. Each of these domains functions an inner ring, and the C-terminal helices form a more as a reversible rotary motor and exchanges energy with loosely packed outer ring (Stock et al., 1999; Vonck et the opposite motor through mechanical rotation of the al. , 2002). In this construction, enough space may be left central stalk . During ATP between neighboring outer helices and the connecting synthesis, the electrochemical ion gradient fuels the inner helix for access channels from the cytoplasm to membrane-embedded F 0 motor to rotate the central the binding sites in the center of the bilayer. stalk in its intrinsic direction. This rotation causes se-Interestingly, the number of c subunits forming the quential binding changes at the peripheral F 1 domain so ring is not fixed but varies among species: numbers of that one catalytic site binds ADP and phosphate, the 10, 11, and 14 have been found for the ATP synthases from yeast, Propionigenium modestum, and spinach chloroplasts, respectively. Hence, there is obviously a

Single Molecule Energetics of F1-ATPase Motor

Biophysical Journal, 2007

Motor proteins are essential in life processes because they convert the free energy of ATP hydrolysis to mechanical work. However, the fundamental question on how they work when different amounts of free energy are released after ATP hydrolysis remains unanswered. To answer this question, it is essential to clarify how the stepping motion of a motor protein reflects the concentrations of ATP, ADP, and P i in its individual actions at a single molecule level. The F 1 portion of ATP synthase, also called F 1 -ATPase, is a rotary molecular motor in which the central g-subunit rotates against the a 3 b 3 cylinder. The motor exhibits clear step motion at low ATP concentrations. The rotary action of this motor is processive and generates a high torque. These features are ideal for exploring the relationship between free energy input and mechanical work output, but there is a serious problem in that this motor is severely inhibited by ADP. In this study, we overcame this problem of ADP inhibition by introducing several mutations while retaining high enzymatic activity. Using a probe of attached beads, stepping rotation against viscous load was examined at a wide range of free energy values by changing the ADP concentration. The results showed that the apparent work of each individual step motion was not affected by the free energy of ATP hydrolysis, but the frequency of each individual step motion depended on the free energy. This is the first study that examined the stepping motion of a molecular motor at a single molecule level with simultaneous systematic control of DG ATP . The results imply that microscopically defined work at a single molecule level cannot be directly compared with macroscopically defined free energy input.

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Energy transduction in the sodium F-ATPase of Propionigenium modestum

Proceedings of the National Academy of Sciences, 1999

The F-ATPase of the bacterium Propionigenium modestum is driven by an electrochemical sodium gradient between the cell interior and its environment. Here we present a mechanochemical model for the transduction of transmembrane sodium-motive force into rotary torque. The same mechanism is likely to operate in other F-ATPases, including the proton-driven F-ATPases of Escherichia coli.

Torque generation by the Fo motor of the sodium ATPase

Biophysical journal, 2004

Based on recent structural and functional findings, we have constructed a mathematical model for the sodiumdriven F o motor of the F 1 F o -ATPase from the anaerobic bacterium Propionigenium modestum. The model reveals the mechanochemical principles underlying the F o motor's operation, and explains all of the existing experimental data on wild-type and mutant F o motors. In particular, the model predicts a nonmonotonic dependence of the ATP hydrolysis activity on the sodium concentration, a prediction confirmed by new experiments. To explain experimental observations, the positively charged stator residue (R227) must assume different positions in the ATP synthesis and hydrolysis directions. This work also illustrates how to extract a motor mechanism from dynamical experimental observations in the absence of complete structural information.

Critical evaluation of the one- versus the two-channel model for the operation of the ATP synthase’s Fo motor

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2000

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.

The motor of the ATP synthase

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1998

A model is presented in which ion translocation through the F 0 part of the ATP synthase drives the rotation of the ring of c subunits (rotor) versus the a subunit (stator). The coupling ion binding sites on the rotor are accessible from the cytoplasm of a bacterial cell except for the c subunit at the interface to the stator. Here, the binding site is accessible from the periplasm through a channel formed by subunit a. In the ATP synthesis mode, a coupling ion is anticipated to pass through the stator channel into the binding site of the adjacent rotor subunit, following the electrical potential. Occupation of this site triggers, probably by electrostatic forces, the rotation of the ring. This makes the binding site accessible to the cytoplasm, where the coupling ion dissociates. Simultaneously, this rotation moves again an empty rotor subunit into the contact site with the stator, where its binding site becomes loaded and rotation continues.

{"__content__"=>"Elastic coupling power stroke mechanism of the F-ATPase molecular motor.", "sub"=>{"__content__"=>"1"}}

Proceedings of the National Academy of Sciences of the United States of America, 2018

The angular velocity profile of the 120° F-ATPase power stroke was resolved as a function of temperature from 16.3 to 44.6 °C using a Δμ = -31.25 at a time resolution of 10 μs. Angular velocities during the first 60° of the power stroke (phase 1) varied inversely with temperature, resulting in negative activation energies with a parabolic dependence. This is direct evidence that phase 1 rotation derives from elastic energy (spring constant, κ = 50 ·rad). Phase 2 of the power stroke had an enthalpic component indicating that additional energy input occurred to enable the γ-subunit to overcome energy stored by the spring after rotating beyond its 34° equilibrium position. The correlation between the probability distribution of ATP binding to the empty catalytic site and the negative values of the power stroke during phase 1 suggests that this additional energy is derived from the binding of ATP to the empty catalytic site. A second torsion spring (κ = 150 ·rad; equilibrium position, 9...

F1FO ATP synthase molecular motor mechanisms

Frontiers in Microbiology

The F-ATP synthase, consisting of F1 and FO motors connected by a central rotor and the stators, is the enzyme responsible for synthesizing the majority of ATP in all organisms. The F1 (αβ)3 ring stator contains three catalytic sites. Single-molecule F1 rotation studies revealed that ATP hydrolysis at each catalytic site (0°) precedes a power-stroke that rotates subunit-γ 120° with angular velocities that vary with rotational position. Catalytic site conformations vary relative to subunit-γ position (βE, empty; βD, ADP bound; βT, ATP-bound). During a power stroke, βE binds ATP (0°–60°) and βD releases ADP (60°–120°). Årrhenius analysis of the power stroke revealed that elastic energy powers rotation via unwinding the γ-subunit coiled-coil. Energy from ATP binding at 34° closes βE upon subunit-γ to drive rotation to 120° and forcing the subunit-γ to exchange its tether from βE to βD, which changes catalytic site conformations. In F1FO, the membrane-bound FO complex contains a ring of...

Sequence of subunit c of the sodium ion translocating adenosine triphosphate synthase of Propionigenium modestum

European Journal of Biochemistry, 1990

The 30 N‐terminal amino acid residues of the purified ATPase c subunit of Propionigenium modestum have been determined. An oligonucleotide mixture was derived from this sequence and used as probe for cloning the corresponding gene in Escherichia coli. The nucleotide sequence of the gene has been determined and compared with those of ATPase c subunits from other bacteria and chloroplasts. Peculiar sequence similarities are found only at the C‐terminus between the c subunits of the ATPases from P. modestum and from Vibrio alginolyticus, another putative Na+‐translocating ATPase.

Structure of the Vacuolar H+-ATPase Rotary Motor Reveals New Mechanistic Insights

Structure, 2015

Graphical Abstract Highlights d Subnanometer V-ATPase EM structure gives new insights into mechanism d Comparison of two distinct catalytic states in a complete rotary ATPase d Describes a conserved electrostatic bearing that supports high motor efficiency d Proposes how different c ring stoichiometries are accommodated

Crucial Role of the Membrane Potential for ATP Synthesis by F1Fo ATP Synthases

Journal of Experimental Biology, 2000

ATP, the universal carrier of cell energy, is manufactured from ADP and phosphate by the enzyme ATP synthase using the free energy of an electrochemical gradient of protons (or Na+). The proton-motive force consists of two components, the transmembrane proton concentration gradient (ΔpH) and the membrane potential. The two components were considered to be not only thermodynamically but also kinetically equivalent, since the chloroplast ATP synthase appeared to operate on ΔpH only. Recent experiments demonstrate, however, that the chloroplast ATP synthase, like those of mitochondria and bacteria, requires a membrane potential for ATP synthesis. Hence, the membrane potential and proton gradient are not equivalent under normal operating conditions far from equilibrium. These conclusions are corroborated by the finding that only the membrane potential induces a rotary torque that drives the counter-rotation of the a and c subunits in the Fo motor of Propionigenium modestum ATP synthase.

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Membrane topography of the coupling ion binding site in Na+-translocating F1F0 ATP synthase

2002

A carbodiimide with a photoactivatable diazirine substituent was synthesized and incubated with the Na ؉translocating F 1 F 0 ATP synthase from both Propionigenium modestum and Ilyobacter tartaricus. This caused severe inhibition of ATP hydrolysis activity in the absence of Na ؉ ions but not in its presence, indicating the specific reaction with the Na ؉ binding c-Glu 65 residue. Photocross-linking was investigated with the substituted ATP synthase from both bacteria in reconstituted 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC)-containing proteoliposomes. A subunit c/POPC conjugate was found in the illuminated samples but no a-c cross-links were observed, not even after ATP-induced rotation of the c-ring. Our substituted diazirine moiety on c-Glu 65 was therefore in close contact with phospholipid but does not contact subunit a. Na ؉ in / 22 Na ؉ out exchange activity of the ATP synthase was not affected by modifying the c-Glu 65 sites with the carbodiimide, but upon photoinduced cross-linking, this activity was abolished. Cross-linking the rotor to lipids apparently arrested rotational mobility required for moving Na ؉ ions back and forth across the membrane. The site of cross-linking was analyzed by digestions of the substituted POPC using phospolipases C and A 2 and by mass spectroscopy. The substitutions were found exclusively at the fatty acid side chains, which indicates that c-Glu 65 is located within the core of the membrane.