The γ-subunit rotation and torque generation in F ₁ -ATPase from wild-type or uncoupled mutant Escherichia coli

Abstract

The rotation of the ␥-subunit has been included in the binding-change mechanism of ATP synthesis͞ hydrolysis by the proton ATP synthase (F O F 1 ). The Escherichia coli ATP synthase was engineered for rotation studies such that its ATP hydrolysis and synthesis activity is similar to that of wild type. A f luorescently labeled actin filament connected to the ␥-subunit of the F 1 sector rotated on addition of ATP.This progress enabled us to analyze the ␥M23K (the ␥-subunit Met-23 replaced by Lys) mutant, which is defective in energy coupling between catalysis and proton translocation. We found that the F 1 sector produced essentially the same frictional torque, regardless of the mutation. These results suggest that the ␥M23K mutant is defective in the transformation of the mechanical work into proton translocation or vice versa.

Subunit rotation of ATP synthase embedded in membranes: a or β subunit rotation relative to the c subunit ring

“…Combined genetic and biochemical studies suggested that the ␥ subunit amino-and carboxyl-terminal domains interact through longrange conformational transmission (9)(10)(11). The ␥ subunit rotation was indicated biochemically by the ␤͞␥ cross-linking (12) and movement of a probe covalently connected to the ␥ subunit (13), and confirmed finally by the continuous rotation of an actin filament connected to the ␥ subunit of the F 1 sector immobilized on a glass surface (14,15). As discussed (15), proton transport through F o should drive ␥ rotation, leading to the ␤ subunit conformational change that releases the product ATP.…”

“…The ␥ subunit rotation was indicated biochemically by the ␤͞␥ cross-linking (12) and movement of a probe covalently connected to the ␥ subunit (13), and confirmed finally by the continuous rotation of an actin filament connected to the ␥ subunit of the F 1 sector immobilized on a glass surface (14,15). As discussed (15), proton transport through F o should drive ␥ rotation, leading to the ␤ subunit conformational change that releases the product ATP. Reversibly, ATP hydrolysis should also drive ␥ rotation, which is transmitted to the F o sector for proton translocation.…”

Rotating proton pumping ATPases: Subunit/subunit interactions and thermodynamics

“…The first high-resolution X-ray structure of the F 1 sector from bovine heart mitochondria clearly showed that the three b subunits in F 1 have different catalytic site conformations (7). A series of single molecule studies (Table 1) on thermophilic Bacillus PS3 and the Escherichia coli enzyme established that the c subunit (8,9) and cec 10 (10-12) assembly in F 1 and F-ATPase (F O F 1 ), respectively, rotate relative to a stator assembly. Thus, the rotation of F-ATPase is coupled with continuous proton transport through a water channel formed from subunits a and c, which include proton translocating residues Arg and Asp (or Glu), respectively (3).…”

Biological application of multi-component nanowires in hybrid devices powered by F1-ATPase motors

“…The initial direct and visual rotation is observed by using a fluorescent actin filament attached to γ subunit (Yasuda et al, 1998), which provides the direct convincing evidence of the rotation of the F 1 -ATPase motor. In addition, other researchers have also observed the rotation of the F 1 -ATPase motor (Omote et al, 1999;Nishio et al, 2002;Pänke et al, 2000). Further, Noji and Yasuda used small colloidal gold bead of 40 nm in diameter as the propeller to study the mechanism of stepping of the F 1 -ATPase motor with highspeed imaging (Yasuda et al, 2001).…”