Microscopic rotary mechanism of ion translocation in the Fo complex of ATP synthases

Abstract

The microscopic mechanism of coupled c-ring rotation and ion translocation in F(1)F(o)-ATP synthases is unknown. Here we present conclusive evidence supporting the notion that the ability of c-rings to rotate within the F(o) complex derives from the interplay between the ion-binding sites and their nonhomogenous microenvironment. This evidence rests on three atomic structures of the c(15) rotor from crystals grown at low pH, soaked at high pH and, after N,N'-dicyclohexylcarbodiimide (DCCD) modification, resolved at 1.8, 3.0 and 2.2 Å, respectively. Alongside a quantitative DCCD-labeling assay and free-energy molecular dynamics calculations, these data demonstrate how the thermodynamic stability of the so-called proton-locked state is maximized by the lipid membrane. By contrast, a hydrophilic environment at the a-subunit-c-ring interface appears to unlock the binding-site conformation and promotes proton exchange with the surrounding solution. Rotation thus occurs as c-subunits stochastically alternate between these environments, directionally biased by the electrochemical transmembrane gradient.

Engineering rotor ring stoichiometries in the ATP synthase

“…As shown in Fig. 5C, the c-subunit stoichiometries revealed in unprocessed topographs include c <11 , c 11 , c 12 , c 13 , c 14 , and c >14 . However, the majority of the c-rings consisted of 11, 12, or 13 subunits (52.6%, 33.3%, and 9.6%, respectively) (Fig.…”

“…A few c-rings in these images were seen to lack individual c-subunits; however, instead of closing this gap so as to form smaller c-rings, these incomplete oligomers have the same shape and diameter as the complete ones (10,25). Furthermore, c-subunits from I. tartaricus and Bacillus TA2.A1 expressed in E. coli were found to assemble correctly into c 11 and c 13 rings, respectively (20,23,24,48), despite the preferred c 10 stoichiometry of the native E. coli c-ring (46). The c-ring sizes also are independent of external factors such as medium pH, host protein expression (48) or host membrane composition (23,24), the source of carbon used by the cell (48,49), and, importantly, the rate of ATP synthesis itself (50,51).…”

“…Based on all F-type ATP synthases known to date, the range spans from n = 8 (9) to n = 15 (10). Each c-subunit contributes to create one binding site for one coupling ion (H + or Na + ) (11,12), which is translocated across the membrane (13). Because the F 1 and F o motors are tightly coupled, the c-ring stoichiometry determines the theoretical ionto-ATP ratio (n/3) of the enzyme, which therefore ranges from 2.7 to 5.…”

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

“…In other words, the a-subunit interacts with two c-subunits, each contacting via a different half channel. The proposed mechanism of proton transfer in the ATP synthesis mode is as follows (37,38,44): a proton enters the half channel exposed to the periplasmic side (or intermembrane space of the mitochondria) and is then transferred to the carboxyl residue of the c-subunit, and transforms to an ion-locked conformation (Fig. 4B) (37).…”

Realistic simulations of the coupling between the protomotive force and the mechanical rotation of the F 0 -ATPase

“…The solution NMR structure (which is based on several assumptions about the a/c interface) indicates that the Asp containing c-ring helix facing the subunit-a is rotated outside forming an open conformation (22). There have been some recent concerns regarding the drastic rotation of the c-ring helix, since such a structural perturbation is inconsistent with the high-resolution crystal structures (9,23). However, the helix rotation upon facing the Arg of subunit-a has been implicated to be important for the functionality of the F 0 motor in other studies (24).…”