Abstract
F-ATP synthases use proton flow through the FO domain to synthesize ATP in the F1 domain. In Escherichia coli, the enzyme consists of rotor subunits γεc10 and stator subunits (αβ)3δab2. Subunits c10 or (αβ)3 alone are rotationally symmetric. However, symmetry is broken by the b2 homodimer, which together with subunit δa, forms a single eccentric stalk connecting the membrane embedded FO domain with the soluble F1 domain, and the central rotating and curved stalk composed of subunit γε. Although each of the three catalytic binding sites in (αβ)3 catalyzes the same set of partial reactions in the time average, they might not be fully equivalent at any moment, because the structural symmetry is broken by contact with b2δ in F1 and with b2a in FO. We monitored the enzyme’s rotary progression during ATP hydrolysis by three single-molecule techniques: fluorescence video-microscopy with attached actin filaments, Förster resonance energy transfer between pairs of fluorescence probes, and a polarization assay using gold nanorods. We found that one dwell in the three-stepped rotary progression lasting longer than the other two by a factor of up to 1.6. This effect of the structural asymmetry is small due to the internal elastic coupling.
Highlights
The F-ATP synthase is a highly flexible and robust motor enzyme [1,2,3,4,5,6,7,8]
In order to correlate structural and rotational asymmetry, we studied actively rotating single EcFO F1 complexes by three distinct single-molecule fluorescence microscopy techniques
The static asymmetric structural elements of F-ATP synthases, of which variations can be found in A-type ATP synthases and V-type ATPases, are as follows: (i) the different conformations and nucleotide occupancies of the three β subunits in the F1 -head at any moment; (ii) the interface of the c-ring with the eccentric subunit a including its two half-channels; (iii) the eccentric position of the peripheral stalk consisting of subunits a, δ, and b2 ; (iv) the interaction between the curved central stalk and each αβ heterodimer at any moment; and (v) the portions of subunits γ and ε that extend beyond the diameter of the c-ring might impose a drag on rotation when passing the peripheral stalk
Summary
The F-ATP synthase is a highly flexible and robust motor enzyme [1,2,3,4,5,6,7,8]. ATP synthesis is coupled to the proton motive force (pmf) or ion motive force across the membrane. Rotation of the c-ring is required to deliver the proton to the other half-channel to complete translocation of the proton across the membrane to the N-side This alternating protonation/deprotonation of c subunits induces a clockwise rotation that is linked to the rotation of the central stalk subunits γ and ε, resulting in ATP synthesis in the catalytic nucleotide-binding sites. During ATP hydrolysis, the γ subunit is forced to rotate CCW and protons are pumped in the opposite direction from the N-side to the P-side, This protonation and deprotonation of the c-ring works like a Brownian ratchet [85,86,87]. In order to correlate structural and rotational asymmetry, we studied actively rotating single EcFO F1 complexes by three distinct single-molecule fluorescence microscopy techniques
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