Siegfried Engelbrecht

ATP SYNTHASE: AN ELECTROCHEMICAL TRANSDUCER WITH ROTATORY MECHANICS

ATP synthase (F0F1-ATPase) uses proton- or sodium-motive force to produce ATP form ADP and P(i). Three lines of experiment have recently demonstrated large-scale intersubunit rotation during ATP hydrolysis by F1. We discuss how ion flow through the membrane-intrinsic portion, F0, may generate torque and how this might be transmitted between stator and rotor to finally expel spontaneously formed ATP from F1 into water.

F-ATPase: specific observation of the rotating c subunit oligomer of EFoEF1

The rotary motion in response to ATP hydrolysis of the ring of c subunits of the membrane portion, Fo, of ATP synthase, FoF1, is still under contention. It was studied with EFoEF1 (Escherichia coli) using microvideography with a fluorescent actin filament. To overcome the limited specificity of actin attachment through a Cys‐maleimide couple which might have hampered the interpretation of previous work, we engineered a ‘strep‐tag’ sequence into the C‐terminal end of subunit c. It served (a) to purify the holoenzyme and (b) to monospecifically attach a fluorescent actin filament to subunit c. EFoEF1 was immobilized on a Ni‐NTA‐coated glass slide by the engineered His‐tag at the N‐terminus of subunit β. In the presence of MgATP we observed up to five counterclockwise rotating actin filaments per picture frame of 2000 μm2 size, in some cases yielding a proportion of 5% rotating over total filaments. The rotation was unequivocally attributable to the ring of subunit c. The new, doubly engineered construct serves as a firmer basis for ongoing studies on torque and angular elastic distortions between F1 and Fo.

ATP synthase: a tentative structural model

Adenosine triphosphate (ATP) synthase produces ATP from ADP and inorganic phosphate at the expense of proton‐ or sodium‐motive force across the respective coupling membrane in Archaea, Bacteria and Eucarya. Cation flow through the intrinsic membrane portion of this enzyme (FO, subunits ab 2 c 9–12) and substrate turnover in the headpiece (F1, subunits α 3 β 3 γ δ ϵ) are mechanically coupled by the rotation of subunit γ in the center of the catalytic hexagon of subunits (α β)3 in F1. ATP synthase is the smallest rotatory engine in nature. With respect to the headpiece alone, it probably operates with three steps. Partial structures of six out of its at least eight different subunits have been published and a 3‐dimensional structure is available for the assembly (α β)3 γ. In this article, we review the available structural data and build a tentative topological model of the holoenzyme. The rotor portion is proposed to consist of a wheel of at least nine copies of subunits c, ϵ and a portion of γ as a spoke, and another portion of γ as a crankshaft. The stator is made up from a, the transmembrane portion of b 2, δ and the catalytic hexagon of (α β)3. As an educated guess, the model may be of heuristic value for ongoing studies on this fascinating electrochemical‐to‐mechanical‐to‐chemical transducer.

Viscoelastic dynamics of actin filaments coupled to rotary F-ATPase: angular torque profile of the enzyme.

ATP synthase (F(O)F(1)) operates as two rotary motor/generators coupled by a common shaft. Both portions, F(1) and F(O), are rotary steppers. Their symmetries are mismatched (C(3) versus C(10-14)). We used the curvature of fluorescent actin filaments, attached to the rotating c-ring, as a spring balance (flexural rigidity of 8. 10(-26) Nm(2)) to gauge the angular profile of the output torque at F(O) during ATP hydrolysis by F(1) (see theoretical companion article (. Biophys. J. 81:1234-1244.)). The large average output torque (50 +/- 6 pN. nm) proved the absence of any slip. Variations of the torque were small, and the output free energy of the loaded enzyme decayed almost linearly over the angular reaction coordinate. Considering the threefold stepping and high activation barrier of the driving motor proper, the rather constant output torque implied a soft elastic power transmission between F(1) and F(O). It is considered as essential, not only for the robust operation of this ubiquitous enzyme under symmetry mismatch, but also for a high turnover rate of the two counteracting and stepping motor/generators.

Inter-subunit rotation and elastic power transmission in F0F1-ATPase.

ATP synthase (F‐ATPase) produces ATP at the expense of ion‐motive force or vice versa. It is composed from two motor/generators, the ATPase (F1) and the ion translocator (F0), which both are rotary steppers. They are mechanically coupled by 360° rotary motion of subunits against each other. The rotor, subunits γϵc 10–14, moves against the stator, (αβ)3δab 2. The enzyme copes with symmetry mismatch (C3 versus C10–14) between its two motors, and it operates robustly in chimeric constructs or with drastically modified subunits. We scrutinized whether an elastic power transmission accounts for these properties. We used the curvature of fluorescent actin filaments, attached to the rotating c ring, as a spring balance (flexural rigidity of 8·10−26 N m2) to gauge the angular profile of the output torque at F0 during ATP hydrolysis by F1. The large average output torque (56 pN nm) proved the absence of any slip. Angular variations of the torque were small, so that the output free energy of the loaded enzyme decayed almost linearly over the angular reaction coordinate. Considering the three‐fold stepping and high activation barrier (>40 kJ/mol) of the driving motor (F1) itself, the rather constant output torque seen by F0 implied a soft elastic power transmission between F1 and F0. It is considered as essential, not only for the robust operation of this ubiquitous enzyme under symmetry mismatch, but also for a high turnover rate under load of the two counteracting and stepping motors/generators.

Domain compliance and elastic power transmission in rotary F(O)F(1)-ATPase.

The 2 nanomotors of rotary ATP synthase, ionmotive FO and chemically active F1, are mechanically coupled by a central rotor and an eccentric bearing. Both motors rotate, with 3 steps in F1 and 10–15 in FO. Simulation by statistical mechanics has revealed that an elastic power transmission is required for a high rate of coupled turnover. Here, we investigate the distribution in the FOF1 structure of compliant and stiff domains. The compliance of certain domains was restricted by engineered disulfide bridges between rotor and stator, and the torsional stiffness (κ) of unrestricted domains was determined by analyzing their thermal rotary fluctuations. A fluorescent magnetic bead was attached to single molecules of F1 and a fluorescent actin filament to FOF1, respectively. They served to probe first the functional rotation and, after formation of the given disulfide bridge, the stochastic rotational motion. Most parts of the enzyme, in particular the central shaft in F1, and the long eccentric bearing were rather stiff (torsional stiffness κ > 750 pNnm). One domain of the rotor, namely where the globular portions of subunits γ and ε of F1 contact the c-ring of FO, was more compliant (κ ≅ 68 pNnm). This elastic buffer smoothes the cooperation of the 2 stepping motors. It is located were needed, between the 2 sites where the power strokes in FO and F1 are generated and consumed.

Two rotary motors in F-ATP synthase are elastically coupled by a flexible rotor and a stiff stator stalk

ATP is synthesized by ATP synthase (FOF1-ATPase). Its rotary electromotor (FO) translocates protons (in some organisms sodium cations) and generates torque to drive the rotary chemical generator (F1). Elastic power transmission between FO and F1 is essential for smoothing the cooperation of these stepping motors, thereby increasing their kinetic efficiency. A particularly compliant elastic domain is located on the central rotor (c10–15/ϵ/γ), right between the two sites of torque generation and consumption. The hinge on the active lever on subunit β adds further compliance. It is under contention whether or not the peripheral stalk (and the “stator” as a whole) also serves as elastic buffer. In the enzyme from Escherichia coli, the most extended component of the stalk is the homodimer b2, a right-handed α-helical coiled coil. By fluctuation analysis we determined the spring constant of the stator in response to twisting and bending, and compared wild-type with b-mutant enzymes. In both deformation modes, the stator was very stiff in the wild type. It was more compliant if b was elongated by 11 amino acid residues. Substitution of three consecutive residues in b by glycine, expected to destabilize its α-helical structure, further reduced the stiffness against bending deformation. In any case, the stator was at least 10-fold stiffer than the rotor, and the enzyme retained its proton-coupled activity.

Cross-linking of engineered subunit delta to (alphabeta)3 in chloroplast F-ATPase

Ser → Cys mutations were introduced into subunit δ of spinach chloroplast F0F1-ATPase (CF0CF1) by site-directed mutagenesis. The engineered δ subunits were overexpressed in Escherichia coli, purified, and reassembled with spinach chloroplast F1-ATPase (CF1) lacking the δ subunit (CF1(−δ)). By modification with eosin-5-maleimide, it was shown that residues 10, 57, 82, 160, and 166 were solvent-accessible in isolated CF1 and all but residue 166 also in membrane-bound CF0CF1. Modification of the engineered δ subunit with photolabile cross-linkers, binding of δ to CF1(−δ), and photolysis yielded the same SDS gel pattern of cross-link products in the presence or absence of ADP, phosphate, and ATP and both in soluble CF1 and in CF0CF1. By chemical hydrolysis of cross-linked CF1, it was shown that δS10C was cross-linked within the N-terminal 62 residues of subunit β. δS57C, δS82C, and δS166C were cross-linked within the N-terminal 192 residues of subunit α. Cross-linking affected neither ATP hydrolysis by soluble CF1 nor its ability to reassemble with CF0 and to structurally reconstitute ATP synthesis. Functional reconstitution, however, seemed to be impaired.

Three-stepped rotation of subunits γ and ϵ in single molecules of F-ATPase as revealed by polarized, confocal fluorometry

The proton translocating ATP synthase is conceived as a rotatory molecular engine. ATP hydrolysis by its headpiece, CF1, drives the rotation of subunit γ relative to the hexagonally arranged large subunits, (αβ)3. We investigated transition states of the rotatory drive by polarized confocal fluorometry (POCOF) as applied to single molecules of engineered, immobilized and load‐free spinach‐CF1. We found that the hydrolysis of ATP caused the stepped and sequential progression of subunit γ through three discrete angular positions, with the transition states of γ being too shortlived for detection. We also observed the stepped motion of ϵ, whereas δ was immobile as (αβ)3.

Functional Halt Positions of Rotary FOF1-ATPase Correlated with Crystal Structures

The F(O)F(1)-ATPase is a rotary molecular motor. Driven by ATP-hydrolysis, its central shaft rotates in 80 degrees and 40 degrees steps, interrupted by catalytic and ATP-waiting dwells. We recorded rotations and halts by means of microvideography in laboratory coordinates. A correlation with molecular coordinates was established by using an engineered pair of cysteines that, under oxidizing conditions, formed zero-length cross-links between the rotor and the stator in an orientation as found in crystals. The fixed orientation coincided with that of the catalytic dwell, whereas the ATP waiting dwell was displaced from it by +40 degrees . In crystals, the convex side of the cranked central shaft faces an empty nucleotide binding site, as if holding it open for arriving ATP. Functional studies suggest that three sites are occupied during a catalytic dwell. Our data imply that the convex side faces a nucleotide-occupied rather than an empty site. The enzyme conformation in crystals seems to differ from the conformation during either dwell of the active enzyme. A revision of current schemes of the mechanism is proposed.