High-resolution single-molecule characterization of the enzymatic states in Escherichia coli F ₁ -ATPase

Abstract

The rotary motor F 1 -ATPase from the thermophilic Bacillus PS3 (TF 1 ) is one of the best-studied of all molecular machines. F 1 -ATPase is the part of the enzyme F 1 F O -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 F 1 -ATPase from Escherichia coli (EF 1 ) is governed by the same mechanism as TF 1 under laboratory conditions. Using optical microscopy to measure rotation of a variety of marker particles attached to the γ-subunit of single surface-bound EF 1 molecules, we characterized the ATP-binding, catalytic and inhibited states of EF 1 . We also show that the ATP-binding and catalytic states are separated by 35±3°. At room temperature, chemical processes occur faster in EF 1 than in TF 1 , 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.

Rotating proton pumping ATPases: Subunit/subunit interactions and thermodynamics

“…Analyzing mutant enzymes with Lys and Cys at position 376 of the a subunit, we concluded that aArg376 does not play a catalytic role, but is important for conformational transmission among the three sites, as discussed in detail previously (5,29). bGlu185 located near aArg376 in the catalytic site is also close to the phosphate moiety of ATP in the crystal structure (17). All substitution mutants of this residue lost the catalytic cooperativity, although they retained the ability of unisite catalysis (30).…”

“…ATP waiting and catalytic dwells were confirmed with the E. coli enzyme (17,18). The rotation of the c 10 ring in F O can be also followed using gold beads, the rate being about $200 rps (M. Sekiya, H. Hosokawa, and M. Futai, unpublished observation).…”

“…The rotation speed is clearly dependent on the viscous drag of the probe attached to the c subunit of F 1 ATPase (13,17). Although actin filament was suitable for demonstrating the rotation of F 1 ATPase, it is desirable to study single molecule rotational catalysis using a probe with minimal load.…”

Elasticity, friction, and pathway of γ-subunit rotation in F o F 1 -ATP synthase

“…The time scale with the three substeps (∼40 ms) agrees well with the experimental values (∼50 ms; ∼60 s −1 for a 120°step) at a maximum load condition (4). This result suggests a possible third substep, yet unresolved, for the E. coli F 1 (34,35). This third step could be associated with phosphate release (31,36).…”

Biophysical Characterization of a Thermoalkaliphilic Molecular Motor with a High Stepping Torque Gives Insight into Evolutionary ATP Synthase Adaptation

“…The two most heavily studied bacterial F 1 ATPases come from the thermophile Bacillus PS3 (TF 1 ) and the mesophile Escherichia coli (EF 1 ). Both enzymes are capable of ATP hydrolysis activity, rotating stepwise at an ATP concentration-dependent rate in an anticlockwise direction (23,40). Because the hexameric ␣ 3 ␤ 3 structure harbors three catalytic sites, the basic step size is 120°coupled to either the consumption or production of one ATP molecule (41).…”