F‐ATPase: specific observation of the rotating c subunit oligomer of EF_oEF₁

Abstract

The rotary motion in response to ATP hydrolysis of the ring of c subunits of the membrane portion, F o , of ATP synthase, F o F 1 , is still under contention. It was studied with EF o EF 1 (Escherichia coli) using microvideography with a fluorescent actin filament. To overcome the limited specificity of actin attachment through a Cys-maleimide couple which might have hampered the interpretation of previous work, we engineered a`strep-tag' sequence into the C-terminal end of subunit c. It served (a) to purify the holoenzyme and (b) to monospecifically attach a fluorescent actin filament to subunit c. EF o EF 1 was immobilized on a Ni-NTA-coated glass slide by the engineered His-tag at the N-terminus of subunit L L. In the presence of MgATP we observed up to five counterclockwise rotating actin filaments per picture frame of 2000 W Wm 2 size, in some cases yielding a proportion of 5% rotating over total filaments. The rotation was unequivocally attributable to the ring of subunit c. The new, doubly engineered construct serves as a firmer basis for ongoing studies on torque and angular elastic distortions between F 1 and F o .

Structural evidence of a new catalytic intermediate in the pathway of ATP hydrolysis by F 1 –ATPase from bovine heart mitochondria

“…During ATP synthesis, a mechanical rotation of the γ-subunit driven by the transmembrane proton-motive force, exerted on a ring of C-subunits in the F o membrane domain of the intact enzyme, takes each of the three β-subunits in its catalytic domain through the states represented by the β TP -, β DP -, and β Esubunits, thereby synthesizing three ATP molecules during each 360°rotation (1,11). Hydrolysis of ATP reverses the direction of rotation, and protons are ejected from the mitochondrial matrix into the intermembrane space through the F o domain of the enzyme (12,13). The rotation of the γ-subunit is not continuous, but it proceeds in 120°steps consisting of 90°and 30°substeps (14).…”

Rotation of the c subunit oligomer in fully functional F1Fo ATP synthase

“…Previous work that showed rotation of the c subunit ring by using fluorescent actin filament technology failed to provide evidence of this. Pänke et al (27) did not measure the effect of F 1 F ospecific inhibitors on their observed rotation of the c subunit ring. Sambongi et al (25) examined the effect of venturicidin, but their results have shown very little inhibition by this potent reagent.…”

“…1B; refs. [25][26][27]. However, none of these studies were conducted with fully functional enzyme because in each case, the F 1 F o being used no longer showed a significant sensitivity to the inhibitors generally used to show that the F 1 F o ATP synthase is coupled (26,28).…”

Stochastic rotational catalysis of proton pumping F-ATPase

“…An actin filament connected to the c ring rotated upon ATP hydrolysis and generated similar torque to that observed for the g rotation . A similar experiment was carried out using a different method to connect the actin probe to the c ring (Pänke et al 2000). Experimental systems for observing rotation were critically discussed Wada et al 2000).…”