The proton-translocating a subunit of F0F1-ATP synthase is allocated asymmetrically to the peripheral stalk

Abstract

The position of the a subunit of the membrane-integral F0 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 epsilon or gamma 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, gamma or epsilon, 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 b2, allowing it to withstand the torque imparted by the rotor during ATP synthesis as well as ATP hydrolysis.

FIGURE 1.

Model of F₀F₁-ATP synthase. Rotating subunits γ, ε, and c₁₀ are colored in black. The static subunits of F₁, α₃β₃δ, are shown in gray. The b subunit dimer is shown as semitransparent gray double tubes, and the possible positions for the membrane-embedded subunit a are shown as a light gray boxes positioned according to cross-linking and stable subcomplex data, that is attached either to the left (gray box labeled A) of the b subunits, between the two b subunits (position B) and the ring of c subunits, or to the right (position C) of b₂.

FIGURE 2.

Photon bursts of single FRET-labeled F₀F₁-ATP synthases upon ATP hydrolysis. Lower panels show fluorescence intensities of FRET donor EGFP fused to subunit a (green trace) and FRET acceptor Alexa568 (red trace) bound to ε56. Upper panels show the calculated proximity factor P (blue trace) with 1-ms time resolution and the mean P value for each assigned FRET level (black line). A, single photon burst recorded with continuous wave excitation at 488 nm. The FRET level transition sequence is L → M → H. B, single photon burst recorded with pulsed excitation at 488 nm. The FRET donor (EGFP) fluorescence lifetimes for the three FRET levels are τ = 2.22 ns (L), τ = 1.87 ns (M), and τ = 0.56 ns (H).

FIGURE 3.

Proximity factor distribution (A) and FRET transition density plot (B) of F₀F₁-ATP synthases during ATP hydrolysis. At least two distinct FRET levels had to be detected within a photon burst to be added to the histograms. A, proximity factors for FRET level L as white bars, for Mas light gray bars, and forHas dark gray bars (825 FRET level in total). B, FRET transition density plot with chromophore distances between EGFP and Alexa568 bound to ε56.

FIGURE 4.

Dwell time distributions of FRET levels L (blue bars, 63 level) (A), M (yellow bars, 247 level) (B), and H (red bars, 71 level) (C) during ATP hydrolysis in the presence of 1 mm ATP are shown. F₀F₁-ATP synthases were labeled with EGFP at the C terminus of a and with Alexa568 at ε56. Dwell times were binned in 5-ms intervals and fitted by monoexponential decays (black curves).

FIGURE 5.

Scheme for allocating the FRET efficiencies during ATP hydrolysis in the model for F₁ when viewed from the membrane. Black arrows pointing to positions I, II, and III indicate the stopping positions of Alexa568 on ε56 (or on γ106) following 120° rotation on a circle with radius ∼2.5 nm (semitransparent gray circular area). The position of the b dimer according to previous FRET measurements (11) is shown as a gray ellipse labeled “b64.” Boxes A and C depict the possible positions of the chromophore in EGFP fused to the C terminus of subunit a.

FIGURE 6.

Individual positions of EGFP (small green balls) according to single molecule FRET triangulation using the FRET pair a-EGFP-ε56-Alexa368 (left side) or a-EGFP-γ106-Alexa568 (right side). The small yellow balls are the individual positions for the FRET acceptor Cy5 at b64 with respect to TMR at ε56 (positions recalculated from previous FRET data of Zimmermann et al. (11)). The γ subunit is shown in red, the ε subunit is in blue. Large light blue balls represent the apparent ε56 stopping positions upon 120° rotation of γε during catalysis. Large orange balls represent the three γ106 positions. Arrows indicate the fluorophore positions at the ε or γ subunit, respectively, for the rotor orientation shown in the images. Upper images show F₁ when viewed from the membrane. In the lower images F₀F₁ are oriented with b₂ subunits to the left side.