Essential arginine residue of the Fo-a subunit in FoF1-ATP synthase has a role to prevent the proton shortcut without c-ring rotation in the Fo proton channel

Abstract

In F(o)F(1) (F(o)F(1)-ATP synthase), proton translocation through F(o) drives rotation of the oligomer ring of F(o)-c subunits (c-ring) relative to F(o)-a. Previous reports have indicated that a conserved arginine residue in F(o)-a plays a critical role in the proton transfer at the F(o)-a/c-ring interface. Indeed, we show in the present study that thermophilic F(o)F(1s) with substitution of this arginine (aR169) to other residues cannot catalyse proton-coupled reactions. However, mutants with substitution of this arginine residue by a small (glycine, alanine, valine) or acidic (glutamate) residue mediate the passive proton translocation. This translocation requires an essential carboxy group of F(o)-c (cE56) since the second mutation (cE56Q) blocks the translocation. Rotation of the c-ring is not necessary because the same arginine mutants of the 'rotation-impossible' (c(10)-a)F(o)F(1), in which the c-ring and F(o)-a are fused to a single polypeptide, also exhibits the passive proton translocation. The mutant (aR169G/Q217R), in which the arginine residue is transferred to putatively the same topological position in the F(o)-a structure, can block the passive proton translocation. Thus the conserved arginine residue in F(o)-a ensures proton-coupled c-ring rotation by preventing a futile proton shortcut.

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

“…As indicated by the map, the positive potential from the Arg ðþÞ residue blocks the protonation from P side and decouples the two proton channels on the P and N side. The role of Arg seems to be consistent with experimental findings (3,11) and also with the implications of some models (12,13,16,17). As the Asp ð−Þ comes close to Arg ðþÞ , the only way for the proton exit is through the N channel, as the proton on the P side faces very high electrostatic barrier.…”

“…Several biochemical and mutational studies have highlighted the role of a conserved Arg residue in mediating the functional ion translocation pathways through the a/c interface. This Arg is located almost at the same position as the H þ ðNa þ Þ binding Asp/Glu residues in the c-ring (10,11). A schematic model of the entire F 0 -ATPase motor is shown in Fig.…”

“…It has then been postulated in a phenomenological way that the directionality of c-ring rotation is enforced by the coupling of the pH-driven proton transport to the protonation/deprotonation events of the Asp residues (12,15). Recent experiments have also suggested that the Arg residues is working to block the uptake of protons from the low pH reservoir (P) once the Asp comes close to the Arg and thus forces the proton to escape to the high pH side (N) (11). However, in spite of the above reasonable assumptions, it is still unclear as to how exactly the pH gradient is tied to the molecular events of protonation and deprotonation of the Asp residues or whether there is an asymmetry in proton transport (PTR), and how this asymmetry can lead to the unidirectional rotation.…”

The ATP Synthase a-subunit of Extreme Alkaliphiles Is a Distinct Variant

“…Thus the observed proton leakiness, which probably accounts both for the malate growth deficit and for the somewhat low and highly variable synthetic capacity at pH 10.5, is not the result of a completely disrupted structure. Rather, the re-positioned Lys-180 may be compromised in a direct role in preventing proton leaks (37) or may be compromised in a role in enhancing the ability of Arg-172 to prevent proton loss through short circuits during rotary function of the enzyme (25,37). In alkaliphiles, specific adaptations of both the c-and a-subunits of the alkaliphile ATP synthase are critical to both the ATP synthesis that achieves this proton capture and to prevention of loss of cytoplasmic protons through the synthase (36,37).…”

“…No high resolution structural data are yet available for the a-subunit, but extensive biochemical and genetic evidence indicates that this ATP synthase subunit plays roles in providing the proton path from outside the membrane surface to the carboxylates of interacting c-subunits of the rotor (4,(17)(18)(19)(20)(21)(22)(23)(24). An essential, conserved arginine in TMH4 (Arg-210 in Escherichia coli) is proposed to prevent proton short-cutting to the cytoplasm without rotation (25) and to cause a shift in the pK a of the essential carboxylate so that the proton that has completed rotation dissociates and enters the proton exit pathway leading to the cytoplasm. That proton exit pathway is also likely to be within the a-subunit (4, 5, 18, 23, 26 -28).…”