Eiro Muneyuki

ATP synthase — a marvellous rotary engine of the cell

ATP synthase can be thought of as a complex of two motors — the ATP-driven F1 motor and the proton-driven Fo motor — that rotate in opposite directions. The mechanisms by which rotation and catalysis are coupled in the working enzyme are now being unravelled on a molecular scale.

Experimental demonstration of information-to-energy conversion and validation of the generalized Jarzynski equality

In 1929, Leo Szilard invented a feedback protocol[? ] in which a hypothetical intelligence called Maxwell’s demon pumps heat from an isothermal environment and transduces it to work. After an intense controversy that lasted over eighty years; it was finally clarified that the demon’s role does not contradict the second law of thermodynamics, implying that we can convert information to free energy in principle[? ? ? ? ? ]. Nevertheless, experimental demonstration of this information-to-energy conversion has been elusive. Here, we demonstrate that a nonequilibrium feedback manipulation of a Brownian particle based on information about its location achieves a Szilard-type information-energy conversion. Under real-time feedback control, the particle climbs up a spiral-stairs-like potential exerted by an electric field and obtains free energy larger than the amount of work performed on it. This enables us to verify the generalized Jarzynski equality[? ], or a new fundamental principle of “information-heat engine” which converts information to energy by feedback control.

Chemomechanical coupling in F1-ATPase revealed by simultaneous observation of nucleotide kinetics and rotation.

F1-ATPase is a rotary molecular motor in which unidirectional rotation of the central γ subunit is powered by ATP hydrolysis in three catalytic sites arranged 120° apart around γ. To study how hydrolysis reactions produce mechanical rotation, we observed rotation under an optical microscope to see which of the three sites bound and released a fluorescent ATP analog. Assuming that the analog mimics authentic ATP, the following scheme emerges: (i) in the ATP-waiting state, one site, dictated by the orientation of γ, is empty, whereas the other two bind a nucleotide; (ii) ATP binding to the empty site drives an ∼80° rotation of γ; (iii) this triggers a reaction(s), hydrolysis and/or phosphate release, but not ADP release in the site that bound ATP one step earlier; (iv) completion of this reaction induces further ∼40° rotation.

Catalysis and rotation of F1 motor: Cleavage of ATP at the catalytic site occurs in 1 ms before 40° substep rotation

F1, a water-soluble portion of FoF1-ATP synthase, is an ATP hydrolysis-driven rotary motor. The central γ-subunit rotates in the α3β3 cylinder by repeating the following four stages of rotation: ATP-binding dwell, rapid 80° substep rotation, interim dwell, and rapid 40° substep rotation. At least two 1-ms catalytic events occur in the interim dwell, but it is still unclear which steps in the ATPase cycle, except for ATP binding, correspond to these events. To discover which steps, we analyzed rotations of F1 subcomplex (α3β3γ) from thermophilic Bacillus PS3 under conditions where cleavage of ATP at the catalytic site is decelerated: hydrolysis of ATP by the catalytic-site mutant F1 and hydrolysis of a slowly hydrolyzable substrate ATPγS (adenosine 5′-[γ-thio]triphosphate) by wild-type F1. In both cases, interim dwells were extended as expected from bulk phase kinetics, confirming that cleavage of ATP takes place during the interim dwell. Furthermore, the results of ATPγS hydrolysis by the mutant F1 ensure that cleavage of ATP most likely corresponds to one of the two 1-ms events and not some other faster undetected event. Thus, cleavage of ATP on F1 occurs in 1 ms during the interim dwell, and we call this interim dwell catalytic dwell.

Evidence for rotation of V1-ATPase

VoV1-ATPase is responsible for acidification of eukaryotic intracellular compartments and ATP synthesis of Archaea and some eubacteria. From the similarity to FoF1-ATP synthase, VoV1-ATPase has been assumed to be a rotary motor, but to date there are no experimental data to support this. Here we visualized the rotation of single molecules of V1-ATPase, a catalytic subcomplex of VoV1-ATPase. V1-ATPase from Thermus thermophilus was immobilized onto a glass surface, and a bead was attached to the D or F subunit through the biotin-streptavidin linkage. In both cases we observed ATP-dependent rotations of beads, the direction of which was always counterclockwise viewed from the membrane side. Given that three ATP molecules are hydrolyzed per one revolution, rates of rotation agree consistently with rates of ATP hydrolysis at saturating ATP concentrations. This study provides experimental evidence that VoV1-ATPase is a rotary motor and that both D and F subunits constitute a rotor shaft.

Pause and rotation of F 1 -ATPase during catalysis

F1-ATPase is a rotary motor enzyme in which a single ATP molecule drives a 120° rotation of the central γ subunit relative to the surrounding α3β3 ring. Here, we show that the rotation of F1-ATPase spontaneously lapses into long (≈30 s) pauses during steady-state catalysis. The effects of ADP-Mg and mutation on the pauses, as well as kinetic comparison with bulk-phase catalysis, strongly indicate that the paused enzyme corresponds to the inactive state of F1-ATPase previously known as the ADP-Mg inhibited form in which F1-ATPase fails to release ADP-Mg from catalytic sites. The pausing position of the γ subunit deviates from the ATP-waiting position and is most likely the recently found intermediate 90° position.

Catalytic Activity of the α3β3γ Complex of F1-ATPase without Noncatalytic Nucleotide Binding Site

A mutant α3β3γ complex of F1-ATPase from thermophilic Bacillus PS3 was generated in which noncatalytic nucleotide binding sites lost their ability to bind nucleotides. It hydrolyzed ATP at an initial rate with cooperative kinetics (Km(1), 4 μM; Km(2), 135 μM) similar to the wild-type complex. However, the initial rate decayed rapidly to an inactivated form. Since the inactivated mutant complex contained 1.5 mol of ADP/mol of complex, this inactivation seemed to be caused by entrapping inhibitory MgADP in a catalytic site. Indeed, the mutant complex was nearly completely inactivated by a 10 min prior incubation with equimolar MgADP. Analysis of the progress of inactivation after initiation of ATP hydrolysis as a function of ATP concentration indicated that the inactivation was optimal at ATP concentrations in the range of Km(1). In the presence of ATP, the wild-type complex dissociated the inhibitory [3H]ADP preloaded onto a catalytic site whereas the mutant complex did not. Lauryl dimethylamineoxide promoted release of preloaded inhibitory [3H]ADP in an ATP-dependent manner and partly restored the activity of the inactivated mutant complex. Addition of ATP promoted single-site hydrolysis of 2′,3′-O-(2,4,6-trinitrophenyl)-ATP preloaded at a single catalytic site of the mutant complex. These results indicate that intact noncatalytic sites are essential for continuous catalytic turnover of the F1-ATPase but are not essential for catalytic cooperativity of F1-ATPase observed at ATP concentrations below ~300 μM.

THERMOPHILIC F1-ATPASE IS ACTIVATED WITHOUT DISSOCIATION OF AN ENDOGENOUS INHIBITOR, EPSILON SUBUNIT

Subunit complexes (α3β3γ, α3β3γδ, α3β3γε, and α3β3γδε) of thermophilic F1-ATPase were prepared, and their catalytic properties were compared to know the role of δ and ε subunits in catalysis. The presence of δ subunit in the complexes had slight inhibitory effect on the ATPase activity. The effect of ε subunit was more profound. The (−ε) complexes, α3β3γ and α3β3γδ, initiated ATP hydrolysis without a lag. In contrast, the (+ε) complexes, α3β3γε and α3β3γδε, started hydrolysis of ATP (<700 μm) with a lag phase that was gradually activated during catalytic turnover. As ATP concentration increased, the lag phase of the (+ε) complexes became shorter, and it was not observed above 1 mm ATP. Analysis of binding and hydrolysis of the ATP analog, 2′,3′-O-(2,4,6-trinitrophenyl)-ATP, suggested that the (+ε) complexes bound substrate only slowly. Differing fromEscherichia coli F1-ATPase, the activation of the (+ε) complexes from the lag phase was not due to dissociation of ε subunit since the re-isolated activated complex retained ε subunit. This indicates that there are two alternative forms of the (+ε) complex, inhibited form and activated form, and the inhibited one is converted to the activated one during catalytic turnover.

V-ATPase of Thermus thermophilus Is Inactivated during ATP Hydrolysis but Can Synthesize ATP

The ATP hydrolysis of the V1-ATPase of Thermus thermophilus have been investigated with an ATP-regenerating system at 25 °C. The ratio of ATPase activity to ATP concentration ranged from 40 to 4000 μm; from this, an apparent K m of 240 ± 24 μm and a V max of 5.2 ± 0.5 units/mg were deduced. An apparent negative cooperativity, which is frequently observed in case of F1-ATPases, was not observed for the V1-ATPase. Interestingly, the rate of hydrolysis decayed rapidly during ATP hydrolysis, and the ATP hydrolysis finally stopped. Furthermore, the inactivation of the V1-ATPase was attained by a prior incubation with ADP-Mg. The inactivated V1-ATPase contained 1.5 mol of ADP/mol of enzyme. Difference absorption spectra generated from addition of ATP-Mg to the isolated subunits revealed that the A subunit can bind ATP-Mg, whereas the B subunit cannot. The inability to bind ATP-Mg is consistent with the absence of Walker motifs in the B subunit. These results indicate that the inactivation of the V1-ATPase during ATP hydrolysis is caused by entrapping inhibitory ADP-Mg in a catalytic site. Light-driven ATP synthesis by bacteriorhodopsin-VoV1-ATPase proteoliposomes was observed, and the rate of ATP synthesis was approximately constant. ATP synthesis occurred in the presence of an ADP-Mg of which concentration was high enough to induce complete inactivation of ATP hydrolysis of VoV1-ATPase. This result indicates that the ADP-Mg-inhibited form is not produced in ATP synthesis reaction.

Thermodynamic efficiency and mechanochemical coupling of F1-ATPase.

F1-ATPase is a nanosized biological energy transducer working as part of FoF1-ATP synthase. Its rotary machinery transduces energy between chemical free energy and mechanical work and plays a central role in the cellular energy transduction by synthesizing most ATP in virtually all organisms. However, information about its energetics is limited compared to that of the reaction scheme. Actually, fundamental questions such as how efficiently F1-ATPase transduces free energy remain unanswered. Here, we demonstrated reversible rotations of isolated F1-ATPase in discrete 120° steps by precisely controlling both the external torque and the chemical potential of ATP hydrolysis as a model system of FoF1-ATP synthase. We found that the maximum work performed by F1-ATPase per 120° step is nearly equal to the thermodynamical maximum work that can be extracted from a single ATP hydrolysis under a broad range of conditions. Our results suggested a 100% free-energy transduction efficiency and a tight mechanochemical coupling of F1-ATPase.