ATP-driven stepwise rotation of F _o F ₁ -ATP synthase

Abstract

FoF1-ATP synthase (FoF1) is a motor enzyme that couples ATP synthesis͞hydrolysis with a transmembrane proton translocation. F1, a water-soluble ATPase portion of FoF1, rotates by repeating ATP-waiting dwell, 80°substep rotation, catalytic dwell, and 40°-substep rotation. Compared with F1, rotation of FoF1 has yet been poorly understood, and, here, we analyzed ATP-driven rotations of FoF1. Rotation was probed with an 80-nm bead attached to the ring of c subunits in the immobilized FoF1 and recorded with a submillisecond fast camera. The rotation rates at various ATP concentrations obeyed the curve defined by a Km of Ϸ30 M and a Vmax of Ϸ350 revolutions per second (at 37°C). At low ATP, ATP-waiting dwell was seen and the kon-ATP was estimated to be 3.6 ؋ 10 7 M ؊1 ⅐s ؊1 . At high ATP, fast, poorly defined stepwise motions were observed that probably reflect the catalytic dwells. When a slowly hydrolyzable substrate, adenosine 5-[␥-thio]triphosphate, was used, the catalytic dwells consisting of two events were seen more clearly at the angular position of Ϸ80°. The rotational behavior of FoF1 resembles that of F1. This finding indicates that ''friction'' in Fo motor is negligible during the ATP-driven rotation. Tributyltin chloride, a specific inhibitor of proton translocation, slowed the rotation rate by 96%. However, dwells at clearly defined angular positions were not observed under these conditions, indicating that inhibition by tributyltin chloride is complex.ATP hydrolysis ͉ binding change mechanism ͉ membrane protein ͉ single-molecule imaging

Temperature Dependence of Single Molecule Rotation of the Escherichia coli ATP Synthase F1 Sector Reveals the Importance of γ-β Subunit Interactions in the Catalytic Dwell

“…This pause is also called the catalytic dwell, which occurs after the 80°and before the 40°subrotation steps, because the reversible hydrolysis/synthesis reaction (k 2 ; Fig. 6, A and B) occurs during this dwell (13,42). The pause duration of the ATP-waiting dwell (after the 40°and before the 80°subrotation steps (19)) is minimized and not resolved in our rotation experiments (Figs.…”

Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes

“…This, however, does not influence the point of maximum activity of V-ATPase (= 2 − 3 mM Na 2 ATP) because at that concentration even 5 mM MgCl 2 can be considered as in excess. Note that 2 mM substrate is considered as high ATP or full speed condition (see, e.g., Yasuda et al 1997;Yasuda et al 2001;Ueno et al 2005;Furuike et al 2011)). We don't know the exact reason of the drop of the activity, also in the case of V-ATPase, at higher substrate concentrations, but one can speculate that, due to the ATP  ADP + Pi dissociation and maybe even ADP impurities in the ATP stock, the ADP concentration might reach a level at which it significantly inhibits ATPases (De la Cruz et al 2000;Nakano et al 2008).…”

“…Crucial for proton transport are the unique glutamic acid residues, one on each subunit c: binding, e.g., dicyclohexyl-carbodiimide to this glutamic acid blocks both proton transport and ATP hydrolysis (Linnett and Beechey 1979;Wada et al 2000;Perez-Sayans et al 2009), proving that catalysis and transport are strongly coupled . In both enzymes this coupling involves a rotation of the rotor relative to the rest of the protein that can be considered as the stator Fillingame et al 2000;Futai et al 2000;Yasuda et al 2001;Rondelez et al 2005;Ueno et al 2005). The rotor vs. stator subunits of V-ATPase are not the same as those of the V o vs. V 1 domains, since subunits a and d of V o belong to the stator, and subunits D and F of V 1 belong to the rotor (other subunits of V o and V 1 belong to the rotor and stator, respectively) (Ubbink- Kok et al 2000).…”

“…(In some cases there are as much as ten-fold differences reported by direct and indirect methods, based on activity measurements on the F-ATPase, even in the very same artificial system, in which the enzyme concentration is known. This discrepancy was attributed to the fraction of as high as 90% inactive F-ATPases (Yasuda et al 2001;Ueno et al 2005;Nakanishi-Matsui et al 2006;Sekiya et al 2009;Nakanishi-Matsui et al 2010;Furuike et al 2011). …”

Operation mechanism of FoF1‐adenosine triphosphate synthase revealed by its structure and dynamics

“…However, little progress has been made owing to the challenges in handling the membrane under an optical microscope and difficulty in stably applying the electrochemical potential high enough to reverse F 1 . Thus far, detergentsolubilized, fully functional F o F 1 was subjected to the rotation assay under ATP hydrolysis conditions (46,47). Single-molecule detection of rotation under ATP synthesis conditions has also been reported, using F€ orster resonance energy transfer (FRET) measurement between two fluorescent dyes conjugated with the stator and rotor subunits of F o F 1 reconstituted into liposomes (48).…”