Synthesis of adenosine triphosphate ATP, the 'biological energy currency', is accomplished by F(o)F(1)-ATP synthase. In the plasma membrane of Escherichia coli, proton-driven rotation of a ring of 10 c subunits in the F(o) motor powers catalysis in the F(1) motor. Although F(1) uses 120 degrees stepping during ATP synthesis, models of F(o) predict either an incremental rotation of c subunits in 36 degrees steps or larger step sizes comprising several fast substeps. Using single-molecule fluorescence resonance energy transfer, we provide the first experimental determination of a 36 degrees sequential stepping mode of the c-ring during ATP synthesis.

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The position of the a subunit of the membrane-integral F 0 sector of Escherichia coli ATP synthase was investigated by single molecule fluorescence resonance energy transfer studies utilizing a fusion of enhanced green fluorescent protein to the C terminus of the a subunit and fluorescent labels attached to specific positions of the ⑀ or ␥ subunits. Three fluorescence resonance energy transfer levels were observed during rotation driven by ATP hydrolysis corresponding to the three resting positions of the rotor subunits, ␥ or ⑀, relative to the a subunit of the stator. Comparison of these positions of the rotor sites with those previously determined relative to the b subunit dimer indicates the position of a as adjacent to the b dimer on its counterclockwise side when the enzyme is viewed from the cytoplasm. This relationship provides stability to the membrane interface between a and b 2 , allowing it to withstand the torque imparted by the rotor during ATP synthesis as well as ATP hydrolysis.F 0 F 1 -ATP synthases are the membrane-embedded rotary enzymes in mitochondria, chloroplasts, and bacteria that provide ATP through oxidative and photophosphorylation (1). In these enzymes, ATP synthesis from ADP and phosphate is driven by the flow of ions, usually H ϩ , down an electrochemical potential difference across the plasma membrane (2). Ion flow through the membrane-integral F 0 sector drives the rotation of the turbine-like ring of c subunits. Extensive analysis of the accessibility of sites within the adjacent a subunit supports the model that this subunit provides two half-channels allowing the proton to access the H ϩ binding site on the c subunit from either side of the membrane (3). Rotation of the c ring by one subunit relative to a is required for net ion translocation by the system. The ␥ and ⑀ subunits of the membrane-peripheral F 1 sector interact with the c ring and turn with it. Rotation of these subunits relative to the catalytic sites housed in the three ␣␤ pairs drive conformational changes that are linked to the binding of substrates as well as the synthesis and release of ATP.The overall structure of the Escherichia coli F 0 F 1 -ATP synthase has been visualized by electron microscopy (4). Single particle analysis and three-dimensional image reconstruction reveal that in addition to the central ␥⑀ stalk connecting F 1 and F 0 a second peripheral stalk links the two sectors. A number of lines of evidence show this peripheral stalk to be composed to two copies of the highly extended b subunit that interact with the single ␦ subunit near the top of the ␣ 3 ␤ 3 hexamer. Because the function of the peripheral stalk is to hold the a subunit and ␣ 3 ␤ 3 stationary to one another while the ␥⑀c 10 rotor turns, it is sometimes called the stator stalk. Although the details of interaction of b 2 ␦ with ␣ 3 ␤ 3 and with the a subunit are unknown, their arrangement and strength must be adequate to withstand the torque imparted by the turning rotor.Limited high resolution structural information is available fo...

Abstract. Catalytic activities of enzymes are associated with elastic conformational changes of the protein backbone. Förster-type resonance energy transfer, commonly referred to as FRET, is required in order to observe the dynamics of relative movements within the protein. Förster-type resonance energy transfer between two specifically attached fluorophores provides a ruler with subnanometer resolution between 3 and 8 nm, submillisecond time resolution for time trajectories of conformational changes, and single-molecule sensitivity to overcome the need for synchronization of various conformations. F O F 1 -ATP synthase is a rotary molecular machine which catalyzes the formation of adenosine triphosphate (ATP). The Escherichia coli enzyme comprises a proton driven 10 stepped rotary F O motor connected to a 3-stepped F 1 motor, where ATP is synthesized. This mismatch of step sizes will result in elastic deformations within the rotor parts. We present a new single-molecule FRET approach to observe both rotary motors simultaneously in a single F O F 1 -ATP synthase at work. We labeled this enzyme with three fluorophores, specifically at the stator part and at the two rotors. Duty cycle-optimized with alternating laser excitation, referred to as DCO-ALEX, allowed to control enzyme activity and to unravel associated transient twisting within the rotors of a single enzyme during ATP hydrolysis and ATP synthesis. Monte Carlo simulations revealed that the rotor twisting is larger than 36 deg.

F o F 1 -ATP synthases catalyze the ATP formation from ADP and phosphate in the membranes of mitochondria, chloroplasts and bacteria. Internal rotation of subunits couples the chemical reaction at the F 1 part to the proton translocation through the F o part. In these enzymes, the membrane-embedded a-subunit is part of the non-rotating 'stator' subunits and provides the proton channel of the F o motor. At present, the relative position of the a-subunit is not known.We examined the rotary movements of the ε-subunit with respect to the non-rotating a-subunit by time resolved singlemolecule fluorescence resonance energy transfer (FRET) using a novel pulsed laser diode. Rotation of the ε-subunit during ATP hydrolysis was divided into three major steps. The stopping positions of ε resulted in three distinct FRET efficiency levels and FRET donor lifetimes. From these FRET efficiencies the position of the FRET donor at the asubunit was calculated. Different populations of the three resting positions of ε, which were observed previously, enabled us to scrutinize the models for the position of the a-subunit in the F o part.