Interactions among γR268, γQ269, and the β Subunit Catch Loop of Escherichia coli F1-ATPase Are Important for Catalytic Activity

Abstract

Removal of the ability to form a salt bridge or hydrogen bonds between the ␤ subunit catch loop (␤Y297-D305) and the ␥ subunit of Escherichia coli F 1 F o -ATP synthase significantly altered the ability of the enzyme to hydrolyze ATP and the bacteria to grow via oxidative phosphorylation. Residues ␤T304, ␤D305, ␤D302, ␥Q269, and ␥R268 were found to be very important for ATP hydrolysis catalyzed by soluble F 1 -ATPase, and the latter four residues were also very important for oxidative phosphorylation. The greatest effects on catalytic activity were observed by the substitution of side chains that contribute to the shortest and/or multiple H-bonds as well as the salt bridge. Residue ␤D305 would not tolerate substitution with Val or Ser and had extremely low activity as ␤D305E, suggesting that this residue is particularly important for synthesis and hydrolysis activity. These results provide evidence that tight winding of the ␥ subunit coiled-coil is important to the rate-limiting step in ATP hydrolysis and are consistent with an escapement mechanism for ATP synthesis in which ␣␤␥ intersubunit interactions provide a means to make substrate binding a prerequisite of proton gradient-driven ␥ subunit rotation.The F 1 F o -ATP synthase 1 uses a non-equilibrium transmembrane proton gradient to catalyze the formation of ATP from ADP and inorganic phosphate. The enzyme consists of two protein complexes, the membrane embedded F o complex, which couples proton translocation to the synthesis of ATP, and the membrane extrinsic F 1 complex, which contains the catalytic sites. The F 1 portion consists of five subunits that occur with a stoichiometric ratio of (␣␤) 3 ␥␦⑀. The ␣ and ␤ subunits are arranged alternately similar to the sections of an orange around a central ␥ subunit stalk (see Fig. 1A). Catalytic activity occurs at the ␣␤ interfaces, primarily within each ␤ subunit. The F 1 portion can be isolated from F o and function as an ATPase (1). The F 1 functions as an ATP driven rotary motor (2), which moves through a complete 360°rotation in three discrete 120°steps. The binding of Mg 2ϩ -ATP to a catalytic site initiates a 90°rotation of the ␥ subunit to form an intermediate state. Following a 2-ms pause, a 30°rotation concurrent with product release completes the catalytic cycle (3).Differences in the conformation of the ␥ subunit have been observed between ground state F 1 structures (4, 5) and the (ADP⅐AlF 4 Ϫ ) 2 F 1 structure by Menz et al. (6), a putative posttransition state structure that contains Mg 2ϩ ADP and SO 4 2Ϫ at the low affinity catalytic site and the transition state analog Mg 2ϩ -ADP-fluoroaluminate bound at the other two catalytic sites. In this intermediate state structure, the position of the ␥ subunit used to attach the probe for rotation studies is rotated approximately 30°from its position in ground state structures. As a consequence, the coiled-coil of the ␥ subunit is more tightly wound in the (ADP⅐AlF 4 Ϫ ) 2 F 1 structure than in the ground state.The major specific interaction between the h...

Load-dependent destabilization of the γ-rotor shaft in F O F 1 ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry

“…This scenario is consistent with the well-established view that torsional elasticity allows γ to serve as an energy reservoir (3,33). The functional significance of strong interactions in the apical bearing is revealed by the findings that disruption of contacts in this region by either truncation of γ or specific amino acid changes to β or γ impairs or prevents ATP-dependent H + -pumping in membrane vesicles and oxidative phosphorylation in vivo (42,43,47).…”

“…The F o F 1 apical bearing region is highlighted in Fig. 5A; it includes the sleeve surrounding the extreme γ C terminus (8), as well as adjacent elements, such as the β-catch loops (42,43). Rotation will be accompanied by steric clashes as γ side chains are forced past the side chains of α 3 β 3 within the tight annular gap of the bearing (15).…”

A Conformational Change of the γ Subunit Indirectly Regulates the Activity of Cyanobacterial F1-ATPase

“…Thus, these residues seem to be important in transferring the conformational change of the ␥ subunit to the catalytic site. In addition, the corresponding mutation analysis in E. coli F 1 showed a drastic decrease in activity (44). As the catch region is formed by ␤ E and ␥ and no nucleotide binds in ␤ E , there are some cooperative interactions among three ␤ subunits as suggested (9).…”

How subunit coupling produces the γ-subunit rotary motion in F 1 -ATPase

“…The third cluster, ␥:252-258 (cyan), is located on the upper part of the C-terminal ␥ helix, making ''catch'' interactions with a ␤ E -subunit loop via salt bridges (␤ E :D316/␥:R254 and ␤ E :D319/ ␥:R254) or hydrogen bonds (␤ E :T318/␥:Q255 and ␤ E :D316/ ␥:Q255) (5). Disruption of the ␤/␥ interactions in the latter region has been shown to yield attenuated activity in both synthesis and hydrolysis (36). The last torque-generation cluster (dark blue) is located at ␥:75-79.…”