Mayumi Nakanishi-Matsui

The mechanism of rotating proton pumping ATPases.

Two proton pumps, the F-ATPase (ATP synthase, FoF1) and the V-ATPase (endomembrane proton pump), have different physiological functions, but are similar in subunit structure and mechanism. They are composed of a membrane extrinsic (F1 or V1) and a membrane intrinsic (Fo or Vo) sector, and couple catalysis of ATP synthesis or hydrolysis to proton transport by a rotational mechanism. The mechanism of rotation has been extensively studied by kinetic, thermodynamic and physiological approaches. Techniques for observing subunit rotation have been developed. Observations of micron-length actin filaments, or polystyrene or gold beads attached to rotor subunits have been highly informative of the rotational behavior of ATP hydrolysis-driven rotation. Single molecule FRET experiments between fluorescent probes attached to rotor and stator subunits have been used effectively in monitoring proton motive force-driven rotation in the ATP synthesis reaction. By using small gold beads with diameters of 40-60 nm, the E. coli F1 sector was found to rotate at surprisingly high speeds (>400 rps). This experimental system was used to assess the kinetics and thermodynamics of mutant enzymes. The results revealed that the enzymatic reaction steps and the timing of the domain interactions among the beta subunits, or between the beta and gamma subunits, are coordinated in a manner that lowers the activation energy for all steps and avoids deep energy wells through the rotationally-coupled steady-state reaction. In this review, we focus on the mechanism of steady-state F1-ATPase rotation, which maximizes the coupling efficiency between catalysis and rotation.

Stochastic high-speed rotation of Escherichia coli ATP synthase F1 sector: The subunit-sensitive rotation

The γ subunit of the ATP synthase F1 sector rotates at the center of the α3β3 hexamer during ATP hydrolysis. A gold bead (40–200 nm diameter) was attached to the γ subunit of Escherichia coli F1, and then its ATP hydrolysis-dependent rotation was studied. The rotation speeds were variable, showing stochastic fluctuation. The high-speed rates of 40- and 60-nm beads were essentially similar: 721 and 671 rps (revolutions/s), respectively. The average rate of 60-nm beads was 381 rps, which is ∼13-fold faster than that expected from the steady-state ATPase turnover number. These results indicate that the F1 sector rotates much faster than expected from the bulk of ATPase activity, and that ∼10% of the F1 molecules are active on the millisecond time scale. Furthermore, the real ATP turnover number (number of ATP molecules converted to ADP and phosphate/s), as a single molecule, is variable during a short period. The ϵ subunit inhibited rotation and ATPase, whereas ϵ fused through its carboxyl terminus to cytochrome b562 showed no effect. The ϵ subunit significantly increased the pausing time during rotation. Stochastic fluctuation of catalysis may be a general property of an enzyme, although its understanding requires combining studies of steady-state kinetics and single molecule observation.

High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F1-ATPase

The rotary motor F1-ATPase from the thermophilic Bacillus PS3 (TF1) is one of the best-studied of all molecular machines. F1-ATPase is the part of the enzyme F1FO-ATP synthase that is responsible for generating most of the ATP in living cells. Single-molecule experiments have provided a detailed understanding of how ATP hydrolysis and synthesis are coupled to internal rotation within the motor. In this work, we present evidence that mesophilic F1-ATPase from Escherichia coli (EF1) is governed by the same mechanism as TF1 under laboratory conditions. Using optical microscopy to measure rotation of a variety of marker particles attached to the γ-subunit of single surface-bound EF1 molecules, we characterized the ATP-binding, catalytic and inhibited states of EF1. We also show that the ATP-binding and catalytic states are separated by 35±3°. At room temperature, chemical processes occur faster in EF1 than in TF1, and we present a methodology to compensate for artefacts that occur when the enzymatic rates are comparable to the experimental temporal resolution. Furthermore, we show that the molecule-to-molecule variation observed at high ATP concentration in our single-molecule assays can be accounted for by variation in the orientation of the rotating markers.

Rotational catalysis in proton pumping ATPases: From E. coli F-ATPase to mammalian V-ATPase

We focus on the rotational catalysis of Escherichia coli F-ATPase (ATP synthase, F(O)F(1)). Using a probe with low viscous drag, we found stochastic fluctuation of the rotation rates, a flat energy pathway, and contribution of an inhibited state to the overall behavior of the enzyme. Mutational analyses revealed the importance of the interactions among β and γ subunits and the β subunit catalytic domain. We also discuss the V-ATPase, which has different physiological roles from the F-ATPase, but is structurally and mechanistically similar. We review the rotation, diversity of subunits, and the regulatory mechanism of reversible subunit dissociation/assembly of Saccharomyces cerevisiae and mammalian complexes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

Single Molecule Behavior of Inhibited and Active States of Escherichia coli ATP Synthase F1 Rotation

ATP hydrolysis-dependent rotation of the F1 sector of the ATP synthase is a successive cycle of catalytic dwells (∼0.2 ms at 24 °C) and 120° rotation steps (∼0.6 ms) when observed under Vmax conditions using a low viscous drag 60-nm bead attached to the γ subunit (Sekiya, M., Nakamoto, R. K., Al-Shawi, M. K., Nakanishi-Matsui, M., and Futai, M. (2009) J. Biol. Chem. 284, 22401–22410). During the normal course of observation, the γ subunit pauses in a stochastic manner to a catalytically inhibited state that averages ∼1 s in duration. The rotation behavior with adenosine 5′-O-(3-thiotriphosphate) as the substrate or at a low ATP concentration (4 μm) indicates that the rotation is inhibited at the catalytic dwell when the bound ATP undergoes reversible hydrolysis/synthesis. The temperature dependence of rotation shows that F1 requires ∼2-fold higher activation energy for the transition from the active to the inhibited state compared with that for normal steady-state rotation during the active state. Addition of superstoichiometric ϵ subunit, the inhibitor of F1-ATPase, decreases the rotation rate and at the same time increases the duration time of the inhibited state. Arrhenius analysis shows that the ϵ subunit has little effect on the transition between active and inhibited states. Rather, the ϵ subunit confers lower activation energy of steady-state rotation. These results suggest that the ϵ subunit plays a role in guiding the enzyme through the proper and efficient catalytic and transport rotational pathway but does not influence the transition to the inhibited state.

Rotational Catalysis of Escherichia coli ATP Synthase F1 Sector STOCHASTIC FLUCTUATION AND A KEY DOMAIN OF THE β SUBUNIT

A complex of γ, ϵ, and c subunits rotates in ATP synthase (FoF1) coupled with proton transport. A gold bead connected to the γ subunit of the Escherichia coli F1 sector exhibited stochastic rotation, confirming a previous study (Nakanishi-Matsui, M., Kashiwagi, S., Hosokawa, H., Cipriano, D. J., Dunn, S. D., Wada, Y., and Futai, M. (2006) J. Biol. Chem. 281, 4126-4131). A similar approach was taken for mutations in the β subunit key region; consistent with its bulk phase ATPase activities, F1 with the Ser-174 to Phe substitution (βS174F) exhibited a slower single revolution time (time required for 360 degree revolution) and paused almost 10 times longer than the wild type at one of the three 120° positions during the stepped revolution. The pause positions were probably not at the “ATP waiting” dwell but at the “ATP hydrolysis/product release” dwell, since the ATP concentration used for the assay was ∼30-fold higher than the Km value for ATP. A βGly-149 to Ala substitution in the phosphate binding P-loop suppressed the defect of βS174F. The revertant (βG149A/βS174F) exhibited similar rotation to the wild type, except that it showed long pauses less frequently. Essentially the same results were obtained with the Ser-174 to Leu substitution and the corresponding revertant βG149A/βS174L. These results indicate that the domain between β-sheet 4 (βSer-174) and P-loop (βGly-149) is important to drive rotation.

Temperature Dependence of Single Molecule Rotation of the Escherichia coli ATP Synthase F1 Sector Reveals the Importance of γ-β Subunit Interactions in the Catalytic Dwell

The temperature-dependent rotation of F1-ATPase γ subunit was observed in Vmax conditions at low viscous drag using a 60-nm gold bead (Nakanishi-Matsui, M., Kashiwagi, S., Hosokawa, H., Cipriano, D. J., Dunn, S. D., Wada, Y., and Futai, M. (2006) J. Biol. Chem. 281, 4126–4131). The Arrhenius slopes of the speed of the individual 120° steps and reciprocal of the pause length between rotation steps were very similar, indicating a flat energy pathway followed by the rotationally coupled catalytic cycle. In contrast, the Arrhenius slope of the reciprocal pause length of the γM23K mutant F1 was significantly increased, whereas that of the rotation rate was similar to wild type. The effects of the rotor γM23K substitution and the counteracting effects of βE381D mutation in the interacting stator subunits demonstrate that the rotor-stator interactions play critical roles in the utilization of stored elastic energy. The γM23K enzyme must overcome an abrupt activation energy barrier, forcing it onto a less favored pathway that results in uncoupling catalysis from rotation.

Stochastic proton pumping ATPases: From single molecules to diverse physiological roles

We discuss the most recent reports on two proton pumps, F‐ATPase (ATP synthase) and V‐ATPase (endomembrane proton pump). They are formed from similar extrinsic (F1 or V1) and intrinsic (Fo or Vo) membrane sectors, and couple chemistry and proton transport through subunit rotation for apparently different physiological roles. Emphasis is placed on the stochastic rotational catalysis of F‐ATPase and isoforms of V‐ATPase. iubmb Life, 58: 318‐322, 2006

Stochastic rotational catalysis of proton pumping F-ATPase

F-ATPases synthesize ATP from ADP and phosphate coupled with an electrochemical proton gradient in bacterial or mitochondrial membranes and can hydrolyse ATP to form the gradient. F-ATPases consist of a catalytic F1 and proton channel F0 formed from the α3β3γδε and ab2c10 subunit complexes, respectively. The rotation of γεc10 couples catalyses and proton transport. Consistent with the threefold symmetry of the α3β3 catalytic hexamer, 120° stepped revolution has been observed, each step being divided into two substeps. The ATP-dependent revolution exhibited stochastic fluctuation and was driven by conformation transmission of the β subunit (phosphate-binding P-loop/α-helix B/loop/β-sheet4). Recent results regarding mechanically driven ATP synthesis finally proved the role of rotation in energy coupling.

ATP synthase from Escherichia coli: Mechanism of rotational catalysis, and inhibition with the ε subunit and phytopolyphenols

ATP synthases (FoF1) are found ubiquitously in energy-transducing membranes of bacteria, mitochondria, and chloroplasts. These enzymes couple proton transport and ATP synthesis or hydrolysis through subunit rotation, which has been studied mainly by observing single molecules. In this review, we discuss the mechanism of rotational catalysis of ATP synthases, mainly that from Escherichia coli, emphasizing the high-speed and stochastic rotation including variable rates and an inhibited state. Single molecule studies combined with structural information of the bovine mitochondrial enzyme and mutational analysis have been informative as to an understanding of the catalytic site and the interaction between rotor and stator subunits. We discuss the similarity and difference in structure and inhibitory regulation of F1 from bovine and E. coli. Unlike the crystal structure of bovine F1 (α3β3γ), that of E. coli contains a ε subunit, which is a known inhibitor of bacterial and chloroplast F1 ATPases. The carboxyl terminal domain of E. coli ε (εCTD) interacts with the catalytic and rotor subunits (β and γ, respectively), and then inhibits rotation. The effects of phytopolyphenols on F1-ATPase are also discussed: one of them, piceatannol, lowered the rotational speed by affecting rotor/stator interactions.